that cell wall modifications within ectomycorrhizal roots arise from cell wall--modifying enzymes of fungal origin.

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

**470T Remodeling of the cell wall of endophytic hyphae of *Epichloë festucae* in the symbiotic interaction with *Lolium perenne*.** N. Noorifar¹, M.S. Savoian¹, T. Weikert², B. Moerschbacher², B. Scott¹ 1) Institute of Fundamental Science, Massey University, Palmerston North, Manawatu, NZ; 2) Institute for Biology and Biotechnology of Plants Westfälische Wilhelms-Universität, Münster, Germany.

*Epichloë festucae* is a filamentous fungus, which forms symbiotic associations with aerial tissues of *Lolium* and *Festuca* grass species. Scanning confocal microscopy (SCM)-based analysis of leaf tissue infiltrated with the chitin-specific molecular probe, WGA-Alexa-488, showed that just septa of endophytic hyphae bound this probe while the entire cell wall was labelled in epiphyllous hyphae. These results suggest that hyphal cell wall chitin is either masked or remodeled in endophytic hyphae. The aims of this project are (i) to test whether *E. festucae* LysM-containing proteins have a role in masking chitin and thereby preventing PAMP-triggered immunity as has been shown for the *Cladosporium fulvum*-tomato pathogen interaction and (ii) to analyze the composition of the cell wall of endophytic and epiphytic hyphae.

Analysis of the *E. festucae* genome identified seven genes encoding LysM domains. The expression of two of these genes, *lymA* and *lymB*, were down-regulated in the transcriptome of three different *E. festucae* symbiosis mutants suggesting they are functionally important in symbiosis. Interestingly, both are divergently transcribed from chitinase-encoding genes that are also down regulated in the symbiosis mutants. To functionally analyse the roles of these genes in symbiosis we have generated *DlymA*, *DlymB*, *DchiA*, *DchiB* single deletion and *DlymA/B* double deletion mutants. Plants singly infected with *DlymA*, *DlymB* or *DchiA* mutants have the same plant-interaction phenotypes as wild type as shown by both SCM and transmission electron microscopy analyses. Phenotypic analyses of the *DchiB* and *DlymA/B* mutants are still in progress. To check fungal cell wall structure we used cell wall component-specific eGFP-based biosensors to understand the differences in structure of *E. festucae* endophytic and epiphytic hyphae. Preliminary results suggest that cell wall chitin is converted to chitosan in endophytic hyphae. This structural change is consistent with a lack of a defence response when *E. festucae* forms a mutualistic symbiotic association with *L. perenne*.

**471F Investigating the circadian clock of *Verticillium dahliae* and its influence on pathogenicity.** *E. Cascant-Lopez*¹,², M. K. Sobczyk¹, L. J. Johnson², R. J. Harrison¹ 1) NIAB/EMR, West Malling, Kent, U.K; 2) University of Reading, Reading, Berkshire, U.K.

Many organisms have evolved to adapt themselves to the rhythmicity of their environment. A broad knowledge of the molecular mechanism of circadian clocks has been gained using the fungal model organism *Neurospora crassa*. Nevertheless, little is known about the existence of circadian clocks in other fungi. As a Sordariomycete, *N. crassa* is related to many important plant pathogens including the vascular wilt *Verticillium dahliae*. We aimed to investigate the presence of a circadian clock in *V. dahliae*, and to study whether the clock influences growth and pathogenicity.

Having identified homologues of the *N. crassa* clock oscillator proteins (FRQ, WC-1, WC-2, FRH, FWD-1 and VVD) in *V. dahliae*, we found high conservation in clock protein domains, which suggested the existence of a functional circadian clock. To characterize the clock system in *V. dahliae*, we studied if the daily formation of conidia and microsclerotia (infective propagules) were regulated in a circadian manner. No evidence for an entrainable, free-running rhythm was observed. This suggests that the morphological phenotype is only a response to cycles in light and temperature. Deleting core clock genes such as *frq* does not have any obvious effect on fungal morphology. Furthermore, temporal gene expression profiling with qRT-PCR in constant darkness showed a lack of rhythmic *frq* expression. However, deletion of the transcription factor/photoreceptor WC-1 prevents ring production in light:dark cycles. Thus, WC-1 is involved in the production of the daily developmental rhythm by mediating transcriptional responses to white light.

To understand the possible loss of clock functionality in *V. dahliae*, we performed an orthology analysis with 29 Sordariomycetes species. We found that among *Verticillium* spp. only *Verticillium alfalfae* had lost an oscillator protein homolog: WC-2. We then investigated the presence of a *N. crassa* Clock Box in the *frq* promoter of *V. dahliae*. The C-Box (5'CGATCCGCT3') is essential in regulating rhythmic expression of *frq* in *N. crassa*. Of all the species tested, only *Magnoporthe poae* presented an identical motif in the *frq* promoter. However, in *Verticillium* spp. a similar motif (5'CGATCCCCT 3') was found, and is present in other related species in the Sordariales. In conclusion, *V. dahliae* presents all the necessary genetic loci for a functional clock, but there is no evidence of rhythmicity in either morphological traits or in gene expression.

**472W Phosphate status-dependent control of interactions with pathogenic and endophytic fungi in *Arabidopsis thaliana*.** K. Hiruma¹,², M. Tanaka¹, L. Tae-Hong¹, N. Kitagawa¹, P. Schulze-Lefert³, Y. Saijo¹,² 1) Biological Sciences, NAIST, Ikoma, Nara, JP; 2) JST, PRESTO, JP; 3) Max Planck Institute for Plant Breeding Reserach, Cologne, Germany.

Plants often rely on root-associated beneficial fungi for uptake of inorganic phosphate (Pi) from soils, while restricting pathogen invasion. It remains elusive how plants modulate root immunity in response to Pi availability, to accommodate beneficial endophytes while retaining effective pathogen resistance. We report alterations in Arabidopsis defense responses against the beneficial root endophyte *Colletotrichum tofieldiae* (*Ct*), which promote plant growth under low Pi conditions and the pathogenic relative of *Ct*, *C.incanum* (*Ci*). Genetic studies combined with RNA-seq analysis reveal that components of an intact plant phosphate starvation response are not only required for *Ct*-mediated plant growth promotion but proper defense responses against *Ct*. Furthermore, damage associated molecular pattern (DAMP) sensing and signaling via PEPRs is required for *Ci* restriction in low Pi conditions. We will present how these two separate pathways regulate the closely related pathogenic and endophytic fungi in low Pi conditions.

**473T Comparative genomics reveals structural variations in the genome of the plant pathogenic fungus *Colletotrichum higginsianum*.** A. Tsushima¹,², P. Gan¹, N. Kumakura¹, M. Narusaka³, Y. Takano⁴, Y. Narusaka³, K. Shirasu¹,² 1) Center for Sustainable Resource Science, RIKEN, Yokohama, Japan; 2) Graduate School of Science, The University of Tokyo, Bunkyo-ku, Japan; 3) Research Institute for Biological Sciences Okayama, Okayama, Japan; 4) Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
The members of the genus *Colletotrichum* cause anthracnose disease on a broad range of crops and have a devastating economic impact. Further, the interaction between *Colletotrichum higginsianum* and its host, the model plant *Arabidopsis thaliana*, has been a useful pathosystem to study fungal hemibiotrophic infection of plants. Therefore, the interaction between *C. higginsianum* and *A. thaliana* has been the focus of many studies on *Colletotrichum* to date. Genomic analyses of other fungal plant pathogens have revealed that fast-evolving genomic compartments are often enriched in genes encoding effector proteins, which are diverse, small, and secreted proteins involved in pathogenicity. In order to investigate genomic variations in *C. higginsianum*, we sequenced the genome of strain MAFF305635 from Japan with PacBio RSII, yielding an assembly of 49.8 Mb consisting of 28 contigs. Then, it was compared to the genome of strain IMI349063 from Trinidad and Tobago (Zampounis et al., 2016). Despite the geographic isolation, the two strains were found to be closely related, sharing 88.2 % of sequence (≥ 99 % identity, ≥ 15 kb). However, this analysis also revealed the presence of extensive genomic rearrangements between the two strains. Among these rearrangements, 6 inter-chromosomal translocations and 4 intra-chromosomal inversions were identified. The whole-genome comparison also revealed the presence of strain-specific regions in the genome of this species. Interestingly, it was found that large-scale genomic rearrangements and strain-specific regions tend to associate with transposable elements. These results suggest that transposable elements may increase the genomic flexibility of this pathogen by contributing to homology-based recombination through their repetitive sequences and alternative translocation. The new insights from our research are expected to accelerate effector candidate gene mining of *C. higginsianum* by considering structural variations in its genome.

**474F** ***Cryptococcus neoformans*** suppresses immune cell infiltration into the brain via a secreted immunomodulatory polysaccharide. S.T. Denham, S. Verma, J.M. Daugherty, R. Reynolds, T.E. Lane, J.C.S. Brown Pathology, University of Utah, Salt Lake City, UT.

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

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

**475W** Evaluation of Synergistic Molecules Discovered from High-throughput Screen. M. Wambaugh, J. Brown Department of Pathology, University of Utah, Salt Lake City, UT.

Antimicrobial resistance is a major problem in the modern world. For systemic fungal infections, antifungal drug resistance only adds to pre-existing challenges: few antifungal targets, severe side effects, and treatments that can last for a year or longer. One strategy to combat resistant fungal infections is treatment with synergistic drug combinations – when two drugs combined have far greater activity than predicted by the sum of each individual activity. These combinations have even been shown to inhibit growth of organisms that are resistant to one of the drugs in the pair. However, systematic identification of synergistic pairs is challenging, as they are relatively rare (4-10% of combinations) and high-throughput screening has been impractical due to scale. We developed a method to identify synergistic drug interactions that is scalable and applicable to a wide range of pathogenic microbes. Using a large-scale chemical genetics dataset from the fungus *Cryptococcus neoformans*, we calculate chemical-genetic signatures – the set of genes whose knockouts are strongly sensitive or resistant to a small molecule – for the antifungal drug fluconazole and its known synergistic partners. We then identify genes whose knockouts respond similarly to all known synergistic partners. We use these "synergy prediction mutants" to screen a collection of FDA approved drugs for synergistic interactions with fluconazole. Our screen is five times faster than testing each combination individually and correctly identifies important interactions 57% of the time. We will present newly identified synergistic pairs identified by our screen, which includes interactions between fluconazole and antidepressants, anticholinergics, and antihistamines. Since *in vitro* synergy does not always translate to efficacy in patients, we also test these combinations in a zebrafish brain model of *Cryptococcus neoformans* infection to further evaluate efficacy. Finally, we investigate the molecular mechanisms underlying these interactions with the ultimate goal of rationally designing synergistic combinations to improve treatment of systemic fungal infections.

**476T** Re-wiring of a conserved stress response pathway connects development, secretion and effector gene expression in *Ustilago maydis*. Kai Heimel¹, Lara Schmitz¹, Florian Finkernagel², Gunther Doehlemann³, Martin Hampel¹ 1) Department of Molecular Microbiology and Genetics, Georg-August-University Goettingen, Germany; 2) Institute of Molecular Biology and Tumor Research, Philipps-University Marburg, Germany; 3) Department of Terrestrial Microbiology, University of Cologne, Germany.

The unfolded protein response (UPR) represents a conserved cellular response to ensure homeostasis of the endoplasmic reticulum (ER). Under ER stress conditions, such as increased demands for protein secretion the UPR is activated and promotes ER expansion and an upregulation of the ER protein folding machinery. In pathogenic fungi a functional UPR is ubiquitously required for virulence, although
the underlying molecular details are not completely resolved, yet. In the corn smut fungus *Ustilago maydis*, the UPR pathway is required for various virulence related processes, including the adaptation to the host environment, execution of developmental transitions and efficient delivery of effector proteins. A direct connection to the signalling pathways controlling development is established through protein-protein interaction between the central UPR regulator Cib1 and Clp1, an essential regulator of fungal development. This interaction triggers fungal proliferation *in planta* and re-shapes UPR signalling for extended activation during pathogenic growth. Moreover, we observed suppressive effects of an active UPR on the MAPK mediated pheromone pathway, preventing developmental transitions at the early stages of the *U. maydis* life cycle. Consistenly, UPR-activation prevents conjugation tube-formation and filamentous growth. By contrast, pathogenic development after plant infection is strictly dependent on an active UPR pathway. Moreover, a functional UPR is critical for secretion of effectors, and directly contributes to transcriptional regulation of genes encoding secreted effectors. Abolishment of the regulatory connection between the UPR and the Pit2 effector did not affect gene expression of *pit2* *in planta*, but resulted in significantly reduced virulence. We therefore hypothesize that the UPR is critical for effector secretion and UPR-dependent transcriptional control of specific effector genes allows for a rapid adaptation of gene expression in response to changing intracellular or environmental conditions.

**477F Similar outcomes but different ways to get there – Diversity of infection development and expression profiles in *Zymoseptoria tritici* isolates.** Janine Haueisen¹,², Christoph Eschenbrenner¹,², Holger Adamiak³, Jonathan Grandaubert¹,²,⁴, Eva H. Stukenbrock¹,² 1) Christian-Albrechts-University of Kiel, Germany; 2) Max Planck Institute for Evolutionary Biology, Ploen, Germany; 3) independent, Kiel, Germany; 4) Institut Curie, Paris, France.

*Zymoseptoria tritici* is a wheat pathogen with a complex, poorly understood hemibiotrophic lifestyle. Although *Z. tritici* is specialized to infect domesticated wheat in putatively uniform agro-ecosystems we observe a high degree of intra-specific diversity. Our aim is to understand the relevance of this diversity during host-pathogen interaction. We conducted infection experiments with *Z. tritici* field isolates from the Netherlands, Denmark and Iran that show similar quantitative virulence phenotypes on the susceptible cultivar Obelisk. Confocal laser scanning microscopy of infected leaves allowed us to characterize four *Z. tritici* "core" infection stages. However, microscopic analysis revealed that during the core infection stages spatial and temporal infection development clearly differs between isolates. In particular, the extent of biotrophic fungal growth in the plant mesophyll varies, ranging from single hyphae limited to the tissue around stomata to dense hyphal networks in the inter-cellular space. We combined the comparative microscopy with stage-specific RNA-seq to capture dynamics in fungal gene expression as well as isolate-specific transcription profiles. Differential gene expression (DGE) analyses showed that transcription in *Z. tritici* isolates is highly dynamic during infection. Expression profiles representing the biotrophic stage are clearly distant from profiles of the necrotrophic stage and up to 7 % of all transcribed genes were identified to be differentially expressed between all four core stages. Moreover, we found these genes to be enriched in genes encoding small secreted proteins indicating that fine-tuned expression of effector candidates is important for infection progress. DGE analyses also revealed that many *Z. tritici* genes have very different expression profiles in the individual isolates. Comparing the stage-specific transcriptomes between the three isolates, we found that up to 20 % of all genes are differentially expressed. In summary, our analyses reveal a core *Z. tritici* infection program but also identify highly diverse infection development, expression profiles and molecular host-pathogen interaction. We conclude that the high levels of intra-species diversity enable rapid adaptations to changes in host and environment and thereby significantly contribute to the success of *Z. tritici* as one of the most damaging pathogens of wheat.

**478W Fungal-bacterial interactions drive community assembly in cheese rind microbiomes.** Benjamin Wolfe, Yuanchen Zhang, Casey Cosetta, Erik Kastman Biology, Tufts University, Medford, MA.

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

**479T Functional ecology of mycobiome shifts associated with plant senescence - linking environmental and experimental re-synthesis metatranscriptomics.** Ko-Hsuan Chen¹, Hui-Ling Liao², Hailee B. Korotkin³, P. Brandon Matheny³, Francois Lutzoni¹ 1) Department of Biology, Duke University, Durham, NC, USA; 2) Soil and Water Sciences Department, University of Florida, Gainesville, FL, USA; 3) Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN, USA.

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

**480F Biochemical, structural, and functional characterization of the Avr4 core effector family in fungi.** Li-Hung Chen¹, Amanda Kohler¹, Anthony Salvucci¹, Nicholas Hurlburt², Benjamin Schwessinger¹, Andrew Fisher²,³, Ioannis Stergiopoulos¹ 1) Plant Pathology, UC Davis, Davis, CA; 2) Chemistry, UC Davis, Davis, CA; 3) Molecular and Cellular Biology, UC Davis, Davis, CA.

Effectors are secreted microbial proteins that facilitate infections. Research in our lab and elsewhere has revealed the presence of abundant homologous core effectors across different fungal species. One such core effector with homologs in many fungal species of Dothideomycete is Avr4, a secreted chitin-binding effector with a conserved CBM14 (carbohydrate-binding module family 14) domain in its structure that was first characterized in the tomato pathogen *Cladosporium fulvum*. Despite their low sequence homology, Avr4 effectors have a similar biological function in protecting fungal cell wall chitin against chitinases of plant and microbial origin. This suggests that next to deregulation of host immunity during infections some effectors can facilitate general fungal fitness and survival. Remarkably, the majority of Avr4 orthologs are still perceived by Cf-4, a trans-membrane receptor-like protein (RLP) from tomato that recognizes Avr4 and mediates resistance. To identify how Cf-4 can perceive so diverse in sequence Avr4 effectors, we have solved the 1.7 Å X-ray crystal structure of *PfAvr4* from the tomato pathogen *Pseudocercospora fuligina* and used site-directed mutagenesis to identify the molecular determinants necessary for ligand-binding and recognition in *PfAvr4*. Our analysis indicated that, in contrast to expectations that the indispensability of the chitin-binding domain (ChtBD) would make it a prime target for recognition by Cf-4, individual chitin-binding residues do not have a direct effect on the *PfAvr4/Cf-4* interaction. This further indicates that the *PfAvr4* ligand-binding function (virulence) is structurally distinct or does not fully overlap with the property of recognition (avirulence). We have also discovered that several Dothideomycetes carry a paralog of Avr4 that we refer to as Avr4-2. Like Avr4, Avr4-2 proteins are small cysteine-rich secreted proteins with a CBM14. The intrinsic function of Avr4-2 is not yet known but preliminary data from biochemical and functional analyses suggest that it has a role during plant infections that is diversified from that of Avr4. Current efforts are concentrated on determining the substrate specificity and biological function of Avr4-2, and obtaining a co-crystal of Avr4 with its chitin substrate in order to define with higher accuracy the molecular basis for the broad recognition of core effectors by cognate immune receptors.

**481W Identification and monitoring of effector proteins in the spinach downy mildew pathogen *Peronospora farinosa*.** Joël Klein¹, Marcel van Verk¹,², Guido van den Ackerveken¹ 1) Plant-Microbe Interactions, Department of Biology, Utrecht University, Utrecht, NL; 2) Bioinformatics, Department of Biology, Utrecht University, Utrecht, NL.

*Peronospora farinosa* f. sp. *spinaciae* (*Pfs*) is an obligate biotrophic oomycete pathogen of spinach, on which it causes downy mildew disease. *Pfs* employs secreted effector proteins to modulate plant innate immunity and enable infection. Resistance genes in the spinach host provide protection against *Pfs*, by recognizing the pathogen, through its effectors. Although newly bred resistant cultivars are initially protected, new *Pfs* races rapidly break the employed resistance genes by adaptation of their recognized effector proteins. A reference genome of *P. farinosa* race 1 (*Pfs1*) of 32 Mbp was generated by sequencing using PacBio, and Illumina technology, followed by a hybrid assembly. Sequencing of *Pfs1* mRNA of 7 infection stages before and during infection contributed significantly to generate accurate gene models. The corresponding protein models have been used to identify more than 60 *Pfs1* effectors, by selecting proteins with signal peptides followed by conserved translocation motifs. To study adaptation of effectors we sequenced 14 other *Pfs* races using Illumina. The genomes of these races were *de novo* assembled, and the effector repertoires determined. Polymorphisms in effector sequences of these races are under study to provide insight into effector evolution. Furthermore, we assembled the mitochondrial genomes of *Pfs1* and the 14 other races to infer their phylogenetic relatedness. Combined with the data on effector evolution this will give us insight into mechanisms by which this downy mildew breaks resistance in spinach cultivars.

**482T Roles of specific *Fusarium graminearum* secreted proteins during Fusarium head blight disease.** A.K. Machado¹, W-S Lee¹, M. Urban¹, F. Olmez², K. Hammond-Kosack¹ 1) Plant Biology Crop Science, Rothamsted Research, Harpenden, United Kingdom; 2) Central Research Institute for Field Crops, Ankara, Turkey.

*Fusarium graminearum* (*Fg*) is a major fungal pathogen of wheat crops globally, causing Fusarium head blight (FHB) disease. Like many other plant pathogens, *Fg* is predicted to produce *in planta* secreted proteins that modulate plant metabolism to suppress or re-programme plant defences. Understanding the molecular functions of *Fg* effectors will help to elucidate the processes underlying wheat spike colonisation and fungal pathogenicity.

With the aim of identifying *Fg* effector proteins that can suppress host plant defences we selected using various criteria a set of small secreted proteins (SSP) to express *in planta* using the *Barley stripe mosaic virus* over-expression system (BSMV-VOX). We then tested whether expression of any of these SSPs enhanced *Fg* fungal infection of susceptible wheat spikes. Amongst the set of *Fg* SSP tested, three (*FgSSP5*, *FgSSP6* and *FgSSP7*) appear to enhance FHB disease when overexpressed in wheat ears prior to infecting with *Fg*.

*FgSSP6* and *FgSSP7* belong to the cerato-platanin protein (CPP) family. In several other plant pathogenic fungi, CPPs have been implicated in a number of virulence and plant protection mechanisms, including induction of host plant cell death or expansin-like activity. *FgSSP5* encodes a protein that possesses the pfam domain RALF (Rapid alkalinization factor; PF05498.6). RALF domain-containing proteins are predominately found in plants and play a role in plant development regulating tissue expansion and/or negatively regulating pollen tube elongation. BLAST analyses identified RALF domain containing proteins in a restrict range of different pathogen species. Based on those results and biochemical tests, our hypothesis is that pre-elevated cerato-platanins (*FgSSP6* and *FgSSP7*) levels in the
apoplast/surrounding the hyphae could induce an intense defence response culminating in cell death to benefit the necrotrophic phase of *Fg* by increasing nutrient availability. FgSSP5 may be a specific virulence factor that manipulates a key plant process, by alkalinizing the plant environment during infection, and using same plant receptor used to recognise plant proteins. Once the mechanisms are further understood, these genes/proteins could potentially be novel intervention targets either for conventional chemistries and/or for methods such as host-induced gene silencing to achieve FHB disease and/or mycotoxin control. The characterisation of single and double gene deletion *Fg* mutants is in progress.

Sponsor: CAPES

**483F** A *SIX1* homolog in *Fusarium oxysporum f.sp. cubense* is required for virulence towards banana. *Sri Widinugraheni*¹,⁵, J.N. Sanchez², H.C van der Does¹, P. van Dam¹, F. Garcia Bastidas³, N. Ordonez³, S. Subandiyah⁴, H.C. Kistler², G.H.J Kema³, M. Rep¹ 1) Molecular Plant Pathology, University of Amsterdam, Amsterdam 904, 1098 XH, NL; 2) ARS USDA, 1551 Lindig Street, St. Paul, MN 551086052, USA; 3) Biointeraction Unit, PRI-WUR, Radix 107 Droevendaalsesteeg 1, Wageningen, NL; 4) Dept. Plant Protection, Gadjah Mada University, Yogyakarta, Indonesia; 5) Fac.of Agriculture, Nusa Cendana University, Kupang, East Nusa Tenggara, Indonesia.

*Fusarium oxysporum f.sp. cubense* (Foc) causes Panama disease in banana. Foc strains are divided into races according to their host specificity. Race 1 infects Gros Michel and Silk varieties, Race 2 infects Bluggoe, a cooking banana variety, and Sub-Tropical Race 4 (ST4) and Tropical Race (TR4) can infect a vast majority of banana varieties, including Cavendish, the variety that is resistant to Race 1 and Race 2.

The *SIX1* effector gene is known to contribute to virulence in *Fusarium oxysporum f.sp. lycopersici* (Fol) and also has an avirulence function by activating the *I*-3 resistance gene of tomato. In Foc, effectors and avirulence factors are still unknown. We found that *SIX1* has three homologs in the Foc Tropical Race 4 (TR4) genome described as *SIX1a/b/c*, of which similarity to Fol SIX1 is 86%, 84% and 82% respectively on the genomic level. At least, *SIX1b* has the highest expression in planta 48 hours after inoculation ¹

To find out which virulence factors are associated with the disease caused by Foc, we chose to first investigate *SIX1a* function by knocking out and complementing the gene. We chose this gene because *SIX1a* is present in all races of Foc. It is also the most similar to Fol *SIX1*, and may therefore be able to activate the *I-3* gene like Fol *SIX1*.

A *SIX1a* deletion mutant of was generated in the Foc TR4 II5 strain. The *SIX1a* gene from II5 strain was then ectopically reinserted in the FocΔ*SIX1a* strain. Disease assays in banana showed that the FocΔ*SIX1a* results in a lower average disease index in comparison to the wild type, and that the virulence of the complementation strains are restored to wild type levels. We conclude that *SIX1a* is required for full virulence of Foc TR4 towards Cavendish banana. We are also investigating whether any of the Foc*SIX1* homologs can induce *I-3* mediated resistance in tomato to reveal a potential avirulence function.

¹ Guo,L., L. Han, L. Yang., et al. (2014). Genome and Transcriptome Analysis of the Fungal Pathogen Fusarium oxysporum f.sp cubense Causing Banana Vascular Disease. Plos One. Vol 9 April (4).

**484W** Biotic and abiotic factors influence the expression of effectors in Leptosphaeria maculans during axenic growth. Michel Meyer¹, Salim Bourras², Julie Gervais¹, Isabelle Fudal¹, Marie-Hélène Balesdent¹, Thierry Rouxel¹ 1) BIOGER, INRA, AgroParisTech, Université Paris-Saclay, Thiverval-Grignon, France; 2) Institute of Plant Biology, University of Zürich, Switzerland.

Plant pathogens secrete effector proteins into host tissues to promote infection through the manipulation of host processes. Sequencing and analyses of the genomes of fungal phytopathogens have shown that they contains tens to hundreds of genes predicted to encode putative effectors. Moreover, global analyses of gene expression revealed that several waves of concerted expression of effector genes take place during host invasion. In sharp contrast with the situation described inplanta, the expression of the effectors is difficult to detect and quantify in axenic cultures because their genes are expressed at a very low level. In the present study, we investigate biotic and abiotic factors that may relieve suppression of expression of effectors during axenic growth. Biotic factors (such as carbon source, nitrate source, antibiotics) as well as abiotic factors (pH, temperature) can influence their expression. Of major interest, incubation of the fungal mycelium with 1ug/ml of an antibiotic of the aminoglycoside family allowed an increase of effector gene expression 20-fold to 60-fold compared to regular axenic growth. An RNAseq analysis aiming at identifying the set of effectors up- and down-regulated in a culture medium supplemented or not with the antibiotic has been performed and results obtained will be presented. This simple system could be a good starting point to characterize the plant signals that trigger fungal effector gene expression.

**485T** Towards understanding the mechanism of cytoplasmic effector translocation during biotrophic development of *Magnaporthe oryzae*. *E. Oliveira Garcia*, B. Valent Plant Pathology, Kansas State University, Manhattan, KS, KS.

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

**486F A highly polymorphic avirulence gene in *Zymoseptoria tritici* induces resistance in wheat.** Lukas Meile, Clemence Plissonneau, Fanny Hartmann, Parvathy Krishnan, Daniel Croll, Bruce McDonald, Andrea Sánchez-Vallet Institut fur Integrative Biologie, ETH, Zurich, CH.

*Zymoseptoria tritici* is a globally distributed pathogen that colonizes wheat plants, causing devastating damages. The fungus has a peculiar infection cycle including a long asymptomatic phase followed by a necrotrophic phase that coincides with a high increase in fungal biomass. Several resistance proteins from specific host cultivars control the development of particular avirulent isolates. These resistance proteins are thought to recognize specific avirulence factors. Until now, only one avirulence gene has successfully been cloned. By means of genetic mapping, we have identified a new avirulence gene. The gene is upregulated upon infection and encodes a cysteine-rich small secreted protein, which indicates that it has a role during infection. Disruption of this gene in the avirulent isolate led to an increase in virulence. Complementation experiments showed that polymorphism in the coding sequence is responsible for the difference in virulence between the two isolates. The avirulence gene is present in all of 131 investigated isolates from across the world and exhibits high sequence polymorphism. The genomic region surrounding the gene has low conserved synteny and only in the virulent isolate two insertions rich in transposable elements are present. Remarkably, in vitro expression of a selection marker under a constitutive promoter is substantially lower when inserted at the position of the avirulence gene compared to a random position in the genome. These data indicate that this region is silenced when the pathogen is not in contact with the plant and that the transposable elements might regulate the expression of the gene. Highly controlled gene expression regulation, high sequence polymorphism and localization in a highly dynamic genomic environment highlight a major role of this avirulence gene in plant colonization.

**487W Characterization of *Magnaporthe oryzae* effectors and their interacting partners in rice.** X. Yan, D.M. Soanes, G.R. Littlejohn, M. Martin Urdiroz, L.S. Ryder, M. Oses-Ruiz, B. Tang, M.J. Kershaw, N.J. Talbot School of Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, GB.

The first line of defense against fungal pathogens by plants is the detection of pathogen-associated molecular patterns by pattern recognition receptors to trigger PAMP-triggered immunity (PTI). Pathogens that overcome PTI and successfully invade host cells do so by delivering effectors that interfere with PTI, leading to effector-triggered susceptibility (ETS). We have identified a family of effector-encoding genes in the rice blast fungus *Magnaporthe oryzae*. We first using RNA-seq to identify genes that were differentially expressed during plant infection and which were temporally co-regulated during the early stages of tissue colonisation by the fungus. The putative effector genes were then characterized by targeted gene deletion and their protein products localized using live-cell imaging. We found a group of apoplastic effectors that outlined invasive hyphae while cytoplasmic effectors accumulated at the biotrophic interfacial complex (BIC). We then identified potential effector targets in rice using yeast two hybrid analysis and co-immunoprecipitation in infected plant tissue. Putative protein-protein interactions were independently confirmed using Bimolecular Fluorescence Complementation (BiFC) assays. In this way, we have characterized a set of 32 *MEP* (*Magnaporthe effector protein*) genes. We will report progress on their characterization and, specifically, the role of *MEP1* and *MEP3* in fungal virulence and host immune suppression, respectively.

**488T The *Sporisorium reilianum* effector Sad1 targets the maize RGLG2-like protein to suppress apical dominance in maize ears.** N. Agrawal, F. Drechsler, M. Romich, J. Schirawski RWTH Aachen University, Applied Microbiology, Aachen, North Rhine Westphalia, DE.

When the biotrophic smut fungus *Sporisorium reilianum* infects maize, it suppresses apical dominance of maize ears. The secreted fungal effector protein Sad1 is responsible for this phenotype but its mechanism of action is unknown. When expressed in *Arabidopsis thaliana*, GFP-Sad1 localizes to the cytoplasm and the nucleus and leads to increased inflorescence branching. From a yeast two-hybrid screen, the maize E3 ubiquitin ligase RGLG2 was identified as one of the strongest interaction partners of Sad1. We show by BiFC that Sad1 interacts with RGLG2 at the plasma membrane in *Nicotiana benthamiana*. RGLG2 has a proposed myristoylation site at its N-terminus. Accordingly, interaction of RGLG2 with Sad1 in yeast two-hybrid experiments was only seen, when the C-terminal end of RGLG2 was used. RGLG2-GFP localized to the plasma membrane when expressed in *Nicotiana benthamiana*, but not when the N-terminal Glycine was mutated to Alanine. In *A. thaliana*, RGLG2 is known to move from the plasma membrane to the nucleus upon stress, and there to function in dampening the stress response. We hypothesize that interaction of Sad1 with RGLG2 interferes with RGLG2 function or transport into the nucleus, which would lead to a prolonged stress response. A prolonged stress response has been suggested before to cause suppression of apical dominance. Hence, Sad1 might lead to suppression of apical dominance by inhibiting the stress response suppression function of RGLG2.

**489F Functional characterization of CRN-like effector proteins in the arbuscular mycorrhiza symbiosis.** S. Walter¹, R. Betz¹, N. Corradi², N. Requena¹ 1) Karlsruhe Institute of Technology, Karlsruhe, DE; 2) University of Ottawa, Canada.

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

**490W Determining the mechanism of action of the Histoplasma capsulatum virulence factor Cbp1.** *D.R. Azimova, B.C. English, A. Sil Microbiology and Immunology, UCSF, San Francisco, CA.*

Intracellular pathogens have developed diverse strategies to evade detection by the immune system and spread effectively through the host. *Histoplasma capsulatum* (*Hc*), an intracellular pathogen of macrophages, is a thermally dimorphic fungus endemic to the Ohio and Mississippi river valleys. The yeast form invades alveolar macrophages and resides in a modified phagolysosome that escapes acidification. In culture, one of most abundant yeast phase-specific proteins that *Hc* secretes is Calcium Binding Protein 1 (*Cbp1*), a critical virulence factor required for apoptosis of macrophages during intracellular infection. *Cbp1* is a 78 amino acid protein with no known calcium-binding domains and no known molecular function. Previous work from our lab showed that *Cbp1* is required for transcriptional induction of pro-apoptotic factors tribbles homolog 3 (TRB3) and C-EBP homologous protein (CHOP) during *Hc* infection, as well as activation of apoptotic executioner caspases 3 and 7 and lysis of host cells. We show here that *Cbp1* localizes to the cytosol during infection of primary murine macrophages using fractionation. This key breakthrough shows, for the first time, that a fungal effector of an intracellular pathogen of humans enters the cytosol during infection. We are currently utilizing a library of *Cbp1* mutants to determine the amino acid residues in *Cbp1* that are necessary for cytosolic localization. Additionally, to characterize the molecular mechanism of *Cbp1* action, we are performing affinity purification of a Strep-tagged allele followed by mass spectrometry (AP-MS). These data will shed light on host-binding partners of *Cbp1* that allow it to modulate macrophage biology.

**491T *Verticillium nonalfalfae* effectors: from –omics approaches to individual function.** *S. Berne, H. Volk, K. Marton, M. Flajšman, V. Progar, B. Javornik* Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.

Plant pathogens express an assortment of effector molecules to modulate plant biological processes and aid infection of specific plant species. Studying effector biology is therefore central to understanding plant-pathogen interactions, including molecular mechanisms of pathogenicity and plant defence responses. For almost a decade, we have been investigating *Verticillium* wilt of hops, predominantly caused by hemibiotrophic sordariomycete *Verticillium nonalfalfae*, using genome sequencing, RNA-Seq and proteomics approaches. Here, we present identification of a repertoire of *V. nonalfalfae* effectors and their subsequent characterization. The *V. nonalfalfae* secretome, comprising 962 putatively secreted proteins, was predicted with an in-house generated pipeline. After a filtering step, 330 candidate secreted effector proteins (CSEPs) with effector-specific domains or without known Pfam domains were obtained and ranked according to various selection criteria (e.g. expression in *planta*). For the 48 best ranked CSEPs, spatio-temporal expression profiling was performed with RT-qPCR in root and shoot samples from infected and control plants, for both susceptible and resistant hop cultivars. Pathogenicity assays in susceptible hop using ATMT-generated effector knock-out mutants revealed 4 CSEPs with reduced virulence and 1 with increased virulence. Two effector proteins *VnaSSP4.2* and *VnaCBP8.213* were selected for functional characterization in biochemical assays. *VnaSSP4.2*, a *Verticillium*-specific small basic protein, was purified from *E. coli* BL21(DE3)pLysS. It bound to multilamellar lipid vesicles in sedimentation assays and formed SDS- and DTT-resistant dimers in POPC:PG, POPC:PE, POPC:SM and POPC:PS vesicles. *VnaCBP8.213*, a carbohydrate binding protein with six hevein domains, was purified from inclusion bodies in *E. coli* SHuffle cells and will be tested for chitin binding, induction of ROS and HR in host and model plants. Unravelling an effector's mode of action may reveal mechanisms essential for *V. nonalfalfae* virulence and provide candidate host proteins that could be exploited in engineering resistance.

**492F *A Phytophthora capsici* virulent RXLR effector targets plant PP2a isoforms that confer *Phytophthora* blight resistance.** *X. Chen*¹,², G. Sheng¹, Y. Li¹, Y. Xing¹, Y. Zhai², W. Ma² 1) College of Horticulture and Plant Protection, Yangzhou University, Yangzhou, Jiangsu Province, China; 2) Department of Plant Pathology and Microbiology, University of California-Riverside, Riverside, California, USA.

Plant pathogens deliver an array of effectors to alter host physiology and defense responses. To understand the molecular basis underlying these, it is important to identify and characterize the target proteins of effectors in plants. Here we show that the RXLR effector *PcAvh1* of *Phytophthora capsici* is highly conserved across *Phytophthora* genus. The encoding gene *PcAvh1* is upregulated during the plant infection stages versus barely expressed during the developmental stages. The effector is important for the pathogen virulence on pepper and *Nicotiana benthamiana* via activity in the host nucleus and cytoplasm and of RNA silencing suppression. *PcAvh1* interacts with two host protein phosphatase 2a (PP2a) structural isoforms. Silencing of the *PP2a* isoforms facilitates pathogen infection. Furthermore, silencing of the isoforms together severely compromises the growth of plants. These results demonstrate that host PP2a activity is required for the plant resistance against the pathogen. Taken together, we conclude that *PcAvh1* is an important virulence determinant that targets host PP2a isoforms to attenuate plant resistance.

**493W Presence of race-specific resistance against oat crown rust in *Brachypodium* species.** *M. Figueroa¹, V. Omidvar¹, S.E. Rottschaefer¹, F. Li¹, D. Singh², R.F. Park², M. Ayliffe³, M. Moscou⁴, P.N. Dodds¹,³, S.F. Kianian¹,⁵* 1) Plant Pathology, University of Minnesota, St.Paul, MN; 2) University of Sydney, Cobbitty, NSW, Australia; 3) CSIRO Agriculture and Food, ACT, Australia; 4) The Sainsbury Laboratory, Norwich NR4 7UH, UK; 5) USDA-ARS, Cereal Disease Laboratory, St. Paul, MN, USA.

*Puccinia coronata* f. sp. *avenae* (Pca), the causal agent of crown rust, represents a serious worldwide agronomic problem in the production of oat (*Avena sativa*). Genetic resistance to control oat crown rust is rather limited; therefore the identification of new resources to reduce yield losses associated with Pca outbreaks is a priority. *Brachypodium* species such as *B. distachyon* and *B. hybridum* show non-host resistance (NHR) against Pca. A collection of *Brachypodium* accessions of these two species was examined to determine the genetic variation for resistance against various North American and Australian Pca pathotypes. Using phenotypic and fungal colonization assays we identified several *Brachypodium* accessions that are ideal for dissecting the genetic architecture controlling NHR against Pca. Our analyses indicate the presence of race specific resistance in both species, highlighting the contribution of effector-triggered immunity to NHR that is likely due to the short evolutionary divergence between oat and *Brachypodium*. Pca isolates with broad virulence in oat showed higher
colonization levels in *Brachypodium* accessions than a Pca isolate with restricted virulence in oat. Transcriptional profiling of several defense-related genes in two *B. distachyon* accessions in response to three Pca isolates during early stages of infection was also conducted. Overall, genes involved in salicylic acid and ethylene signaling pathways were upregulated during the first 48 h after infection. Interestingly, a hexose transporter gene, *BdSTP13*, was also upregulated, which is deemed to be a putative susceptibility factor to wheat rusts. The amplitude of transcriptional upregulation of these genes correlated with rust colonization levels. A study of the genetic basis of resistance to Pca has been initiated in order to discover the factors governing recognition of Pca in *B. distachyon* and to provide foundational knowledge to direct genetic engineering programs in oat improvement.

**494T The discovery of the virulence gene ToxA in the wheat and barley pathogen Bipolaris sorokiniana.** Megan C. McDonald¹, Dag Ahren², Steven Simpfendorfer³, Andrew Milgate⁴, Peter S. Solomon¹ 1) Plant Science, The Australian National University, Canberra, ACT, Australia; 2) Department of Biology, Lund University, Lund, Sweden; 3) NSW Department of Primary Industries Tamworth Agricultural Institute, Tamworth, NSW, Australia; 4) NSW Department of Primary Industries Wagga Wagga Agricultural Institute, Wagga Wagga, NSW, Australia.

*Bipolaris sorokiniana* is the causal agent of multiple diseases on wheat and barley and is the primary constraint to cereal production throughout South Asia. Despite its significance, the molecular basis of disease is poorly understood. To address this, the genomes of three Australian isolates of *B. sorokiniana* were sequenced and screened for known pathogenicity genes.

Sequence analysis revealed that the isolate BRIP10943 harboured the *ToxA* gene which was previously associated with disease in the wheat pathogens *Parastagonospora nodorum* and *Pyrenophora tritici-repentis*. The proteinaceous necrotrophic effector, ToxA, was first identified in culture filtrates of the fungal wheat pathogen *Pyrenophora tritici-repentis* where the culture filtrate alone caused necrotic lesions on susceptible wheat cultivars, in a genotype-specific manner. Later, a near identical 11kb region containing ToxA was found in the genome sequence of the wheat pathogen *Parastagonospora nodorum*. Susceptibility of wheat to ToxA was eventually linked to the presence of a host immune-like gene, *Tsn1*.

Analysis of the regions flanking ToxA within *B. sorokiniana* revealed that it was embedded within a 12kb genomic element nearly identical to the corresponding regions in *P. nodorum* and *P. tritici-repentis*. A screen of 35 Australian *B. sorokiniana* isolates confirmed that ToxA was present in 12 isolates. Sequencing of the ToxA genes within these isolates revealed two sequence haplotypes, which differ by a single non-synonymous nucleotide substitution. Pathogenicity assays show a *B. sorokiniana* isolate harbouring ToxA is more aggressive on wheat lines that contain the sensitivity gene when compared to a non-ToxA isolate. Based on available data, we speculate on the evolutionary origins of this gene in these three fungal wheat pathogens. We propose that ToxA and its surrounding 12kb is a highly promiscuous genomic element, that has the ability to cross species barriers to facilitate disease on wheat. Acquisition of this element can dramatically increase the fitness of pathogenic strains, which in an agricultural setting can have devastating economic and social impacts.

**495F Structural and functional characterization of Leptosphaeria maculans effectors: the example of AvrLm4-7.** Yohann Petit-Houdenot¹, Françoise Blaise¹, Clémence Plissonneau¹, Thierry Rouxel¹, Marie-Hélène Balesdent¹, Karine Blondeau², Noureddine Lazar², Inès Gallay², Théo Le Moigne², Herman van Tilbeurgh², Isabelle Fudal¹ 1) INRA/AgroParisTech UMR1290 BIOGER, French National Institute for Agronomical Research, Thiverval-Grignon, FR; 2) UMR I2BC Université Paris-Sud / CNRS / CEA, Orsay, France.

During plant infection, pathogens secrete an arsenal of effectors, key elements of pathogenesis which modulate innate immunity of the plant and facilitate infection. Fungal effector genes typically encode small proteins, predicted to be secreted, with no homology in databases, and absence of known motif. As such their function or role in pathogenesis is mostly unknown. The phytopathogenic ascomycete *Leptosphaeria maculans* is the causal agent of stem canker of oilseed rape. More than 650 putative effector-encoding genes have been identified in its genome, 7 of them corresponding to avirulence proteins. We develop a project aiming at elucidating the involvement of *L. maculans* effectors in pathogenicity through the structural and functional characterization of a few major effector proteins and the determination of their interactants.

Our strategy is illustrated here with AvrLm4-7, a 143 amino-acid long secreted protein important for fungal fitness and recognized by two oilseed rape resistance proteins, Rlm4 and Rlm7. One single amino-acid change is sufficient to lose recognition by *Rlm4* while maintaining recognition by *Rlm7*. 3D-structure of an isoform of AvrLm4-7 only recognized by Rlm7 was previously determined, allowing us to define regions implicated in recognition by Rlm7 and translocation into plant cell. We recently determined the 3D-structure of another isoform of AvrLm4-7 recognized both by Rlm4 and Rlm7, showing that the amino-acid change allowing to escape *Rlm4*-recognition was located on an external loop and did not change the overall structure of the protein. AvrLm4-7 was also recently shown to suppress recognition of another *L. maculans* avirulence gene, *AvrLm3*, by its cognate resistance gene *Rlm3*, leading us to hypothesize a suppression of Effector-Triggered Immunity (ETI) by AvrLm4-7. In order to test that hypothesis, we transiently expressed AvrLm4-7 and several cell-death inducers in *Nicotiana benthamiana* epidermal cells: AvrLm4-7 was able to suppress cell death induced by BAX and AvrPto. We also generated transgenic lines of *Arabidopsis thaliana* constitutively expressing AvrLm4-7 and are currently characterizing the lines for their susceptibility to pathogens with contrasted lifestyles and for their ability to suppress recognition of *Pseudomonas syringae* avirulence proteins. A better understanding of the role of an effector implicated in the masking of another effector will allow us to develop alternative strategies to genetically control stem canker disease.

**496W The mode of action and origin of RNase-like effectors in powdery mildew fungi.** Pietro Spanu¹, Linhan Li¹, Michal Przydacz¹, Summer Zhang¹, Helen Pennington², Laura Davis³, Ian Dry³ 1) Department of Life Sciences, Imperial College London, London, United Kingdom; 2) The Sainsbury Laboratory, Norwich, United Kingdom; 3) CSIRO Plant Industry, Australia.

The fungi causing powdery mildews on cereals and other plants possess extraordinarily large repertoires of proteins with typical features of pathogenicity effectors. The most prominent of these are a large superfamily of proteins that resemble canonical RNases: the RNAse-Like Proteins associated with Haustoria: the “RALPH” effectors. Here, we provide evidence that RALPH effectors interfere with host immune responses that target ribosomal RNA. RALPH effectors are highly expressed in the plant host and are subject to high rates of selective pressure to diversify; this suggests that plants adopt mechanisms for recognising RALPH effectors which, in turn, drives effector
diversification. In the last year, several seminal publications have demonstrated that, as predicted, cereal immune receptors encoded by resistance (R) genes recognise RALPH effectors. RALPH effectors are present in all powdery mildew fungi analysed so far. We report here that RALPH effectors appear to have evolved from an ancestral *ur*-RALPH. The *ur*-RALPH effector was likely to have been crucial to the early evolution of powdery mildew fungi in the Cretaceous at least 70 MYr ago.

**497T The role of *Phytophthora infestans* effector Avr1-like in modulating plant defense.** R.R. Vetukuri¹, P. B. Kalyandurg², P Boevink³, S. C. Whisson³, E. I. Savenkov², L Grenville-Briggs¹ 1) Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, SE; 2) Department of Plant Biology, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden; 3) Cell and Molecular Sciences, The James Hutton Institute, Dundee, UK.

*Phytophthora infestans* secretes many RXLR effectors potentially involved in infection of host plants. *Avr1-like* is one such RXLR effector that acts as a virulence factor, promoting colonization. Avr1-like is closely related to Avr1 but does not trigger R1-mediated resistance. Unlike Avr1, Avr1-like is present in all the modern European isolates tested in our study. High numbers of transcripts encoding *Avr1-like* were present throughout infection stages, suggesting that this gene is specifically induced in contact with host tissue to modulate plant defences, as seen with other known avirulence effectors such as *Avr3a* and *Avr2*. In this study we demonstrate that *Avr1-like* acts as a suppressor of RNA silencing. Using mutagenesis analysis, we found that a motif in Avr1-like that plays a major role in its suppression activity. Subcellular localization of AVR1 in *Nicotiana benthamiana* by transient expression revealed that this RXLR effector was localized to both the nucleus and the cytoplasm.

**498F In planta localisation of *Leptosphaeria maculans* effectors and identification of their plant targets.** C. Marais¹, Y. Petit¹, F. Blaise¹, B. Ollivier¹, J. Gervais¹, T. Le Moigne², T. Rouxel¹, M.H. Balesdent¹, I. Gallay², K. Blondeau², N. Lazar², H. van TilBeurgh², I. Fudal¹ 1) BIOGER, INRA, Thiverval-Grignon, FR; 2) UMR I2BC, Université Paris-Sud, CNRS, CEA, Orsay, FR.

Fungal effector genes are very diverse and typically encode small proteins, predicted to be secreted, with no or low homology in databases, and absence of known motif. As such their function or role in pathogenesis is mostly unknown. On these bases, the StructuraLEP project aims at elucidating the involvement of *L. maculans* effectors in pathogenicity through the structural and functional characterization of a few major effector proteins and the determination of their interactants. We are investigating six *L. maculans* effectors chosen for their biological significance (involvement in fungal fitness, cognate R gene identified) or because they may represent novel modes of interaction with their plant target (two AVR genes have to be recognized by a specific R gene). We present here the strategies developed within the project to answer two questions: (i) "Where do *L. maculans* effectors act during plant infection?" and (ii) "Which proteins interact with *L. maculans* effectors?". In order to localise *L. maculans* effectors into plant cells and to identify their plant targets, we will transiently express effectors with a fluorescent tag into tobacco leaf epidermal cells and observe effector localisation by confocal microscopy. Tobacco leaves expressing effectors will be used to perform pull-down assays and tobacco proteins interacting with effectors will be identified through mass spectrometry. A more exploratory but biologically relevant strategy will also be tested. *L. maculans* transformants stably expressing effectors with HA-tag will be used to infect oilseed rape leaves. Localisation of effectors in oilseed rape leaves will be performed by immunocytolocalisation using antibodies against the HA-tag. The infected oilseed rape leaves will also be used to perform pull-down assays in order to identify plant proteins targeted by *L. maculans* effectors.

**499W Characterization of effector candidates from the soybean rust fungus that suppress plant immunity.** M. Qi¹, T Link², R Voegele², T Baum¹, S Whitham¹ 1) Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA; 2) Institut für Phytomedizin, Universität Hohenheim, Otto-Sander-Straße 5 70599 Stuttgart, Germany.

Soybean rust (SBR) is an important disease caused by *Phakopsora pachyrhizi* (*Pp*). Like other rust fungi, *Pp* forms specialized cells called haustoria, which are key hubs of molecular exchange with host cells. Haustoria express effector proteins that are transferred to plant cells where they promote fungal growth. We have recently sequenced the *Pp* haustorial transcriptome and predicted candidate effector proteins. To gain insights into functions of *Pp* effector candidates (*PpECs*), we have cloned 82 *PpEC* coding sequences and delivered the proteins into host and non-host plants using a bacterial type III secretion system-based delivery vector. Several *PpECs* suppressed immune responses associated with effector- and pattern-triggered immunity, and we are in the process of characterizing these further. One interesting example is *PpEC23*, a small, cysteine-rich effector candidate that attenuated the hypersensitive response caused by *Pseudomonas syringae* pv *tomato* DC3000 on soybean and tobacco, and it also suppressed basal defense on tobacco and Arabidopsis. *PpEC23* interacts with a transcription factor that our data suggests negatively regulates soybean defenses. We are now using host-induced gene silencing and transcriptomics to determine the effects of *PpEC23* and the interacting soybean transcription factor in immunity or virulence. The results are expected to provide mechanistic insights into the molecular basis of SBR and may be relevant to other rust diseases.

**500T The poplar rust fungus effector biology: challenges of functional characterization of effectors in a non-model pathosystem.** C. Lorrain¹, Benjamin Petre¹, Diane Saunders², Jan Sklenar³, Joe Win³, Sophien Kamoun³, Christine Delaruelle¹, Jérémy Petrosky¹, Pascal Frey¹, Arnaud Hecker¹, Sébastien Duplessis¹ 1) UMR 1136 IAM, Centre INRA Nancy-Lorraine, Champenoux, Lorraine, FR; 2) Earlham Institute, Norwich Research Park, Norwich, United Kingdom; 3) The Sainsbury Laboratory, Norwich Research Park, Norwich, United Kingdom.

Rust fungi are devastating biotrophic pathogens manipulating host processes by delivering effector proteins into the plant cells. The poplar leaf rust fungus *Melampsora larici-populina* genome analysis revealed a large set of secreted proteins that some have been considered as candidate effectors. The understanding how these effector proteins function in the host cells has been the key question of effector biology for the last decade. Many efforts have been made in the field plant-microbe molecular interactions to unravel their role in the colonization of plant tissues. The poplar-poplar rust pathosystem although considered, as a genomic model in the study of tree-microbe interactions is actually non-model pathosystem when it comes to functional characterization of effectors. Absence of easy-going transformation systems for poplar and rust fungi is a major drawback. However, we combined several tools and approaches to help at elucidate the roles of *M. larici-populina* effector proteins such as heterologous *in planta* expression, recombinant protein production or structural approach. All these diverse approaches have led to partially unravelling the role of numerous *M. larici-populina* effector
candidates. Summary report of the ongoing research aimed at elucidating candidate effectors functions will be presented, rising questions about the methodological limits of the study of effector biology in non-model pathosystems.

**501F An insight into necrotrophic effector gene regulation and host-specific virulence of two major Pleosporales fungal pathogens of wheat.** Kasia Rybak, Huyen Phan, Pao Theen See, Robert Syme, Caroline Moffat, Richard Oliver, Kar-Chun Tan Centre for Crop Disease Management, Department of Environment and Agriculture, Curtin University, Bentley, WA 6102, Australia.

The fungus *Parastagonospora nodorum* is the causal agent of septoria nodorum blotch (SNB) of wheat (*Triticum aestivum*). The pathosystem is mediated by multiple fungal necrotrophic effector-dominant host sensitivity gene interactions. The three best characterised effector-sensitivity gene systems are *SnToxA*, *Tsn1*, *SnTox1-Snn1* and *SnTox3-Snn3*. These necrotrophic effector genes are highly expressed during early infection. However, it is not known how these necrotrophic effector genes are regulated in *P. nodorum* until recently. We identified and characterised a gene, referred to as *PnPf2*, which encodes a putative zinc finger transcription factor. *PnPf2* is expressed maximally during early infection like *SnToxA*, *SnTox1* and *SnTox3*. *PnPf2* deletion resulted in down-regulation of *SnToxA* and *SnTox3* expression. Virulence on *Tsn1* and *Snn3* wheat was highly reduced. *SnTox1* expression and virulence on *Snn1* wheat remained unaffected in the deletion background. We also determined that *SnTox1-Snn1* is epistatic to the *SnTox3-Snn3* interaction during SNB. The mechanism of epistasis can be explained - in part - by the suppression of *SnTox3* expression on the presence of *SnTox1*. However, *SnTox1* deletion in the *pnpf2* background did not restore *SnTox3* expression. This indicates that *PnPf2* is dominant over *SnTox1*-mediated epistasis of *SnTox3*. We have also identified and deleted an orthologous *Ptrf2* from the tan spot fungus *Pyrenophora tritici-repentis* which possesses a near-identical *ToxA* that was acquired from *P. nodorum* via horizontal gene transfer. *Ptrf2* deletion also resulted in the down-regulation of *PtrToxA* expression and a near-complete loss of virulence on wheat. We have demonstrated, for the first time, evidence of a functionally conserved signalling component that plays a role in the regulation of common/horizontally transferred effector found in two major pathogens of wheat.

**502W Functional analysis of *Epichloë festucae* small secreted proteins in the symbiotic interaction with *Lolium perenne*.** B. Hassing¹, C. H. Mesarich², C. J. Eaton¹, B. Scott¹ 1) Institute of Fundamental Science, Massey University, Palmerston North, Manawatu, NZ; 2) Institute of Agriculture and Environment, Massey University, Palmerston North, Manawatu, NZ.

*Epichloë* endophytes form stable and mainly mutually beneficial associations with a wide range of cool-season grasses. In a transcriptome analysis, 14 genes encoding small secreted proteins (SspB-SspO) were identified as differentially expressed in four different symbiosis-deficient strains of *E. festucae* during *in planta* colonization of *Lolium perenne*¹. Many SspS, also called effectors, have been shown to interfere with the host defence response in pathogenic and symbiotic interactions. All 14 putative effectors were analysed with bioinformatic tools, and from these, four, specifically SspL, SspM, SspN and SspO, were chosen for further functional analysis. Their secretion was verified using a secretion signal-trap assay and Western analysis. The deletion of *sspL*, *sspM*, *sspN* and *sspO* did not result in an obvious whole-plant interaction phenotype distinguishing WT- and deletion mutant-infected plants, except for a slight increase in anthocyanin levels in the *sspO* deletion strain infected plants. Furthermore, the analysis of infected plant tissue with confocal microscopy and TEM did not identify any obvious differences in cellular phenotype. Currently the effect of Ssp overexpression on whole-plant interaction and cellular phenotypes is being explored. In addition, Ssp localization studies are being conducted to gain further information about the function of these proteins *in planta*. In the future the effect of the Ssp on *L. perenne* gene expression will be analysed and *in planta* interaction partners will be identified.

¹Eaton C. J., Dupont P. Y., Solomon P., Clayton W., Scott B., Cox M. P. (2015). A Core Gene Set Describes the Molecular Basis of Mutualism and Antagonism in *Epichloë spp.* Mol Plant Microbe Interact. 28:69-85.

**503T An antimicrobial and phytotoxic ribonuclease secreted by the wheat pathogen *Zymoseptoria tritici*.** G.J. Kettles, C. Bayon, C.A. Sparks, K. Kanyuka, J.J. Rudd Plant Biology & Crop Science, Rothamsted Research, Harpenden, Hertfordshire, UK.

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

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

**504F Functional characterization of a protein complex formed by four *Ustilago maydis* effectors essential for virulence.** N. Ludwig¹, L. Liang¹, K. Schipper¹,², S. Reißmann¹, D. Aßmann¹, T. Glatter¹, J. Altmüller³, R. Kahmann¹ 1) Max Planck Institute for Terrestrial Microbiology, Marburg, Germany; 2) Heinrich Heine University Düsseldorf, Düsseldorf, Germany; 3) Cologne Center for Genomics (CCG), Cologne University, Cologne, Germany.

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

**505W Identification of effector candidates for avirulence genes in the wheat stripe rust fungus (Puccinia striiformis f. sp. tritici) by secretome analysis.** C. Xia¹, M. Wang¹, O. Cornejo², D. Jiwan¹, D. See¹, X Chen¹,³ 1) Department of Plant Pathology, Washington State University, Pullman, WA; 2) School of Biological Sciences, Washington State University, Pullman, WA; 3) USDA, ARS, Pullman, WA.

*Puccinia striiformis* f. sp. *tritici* (*Pst*) causes stripe rust (yellow rust, *Yr*), one of the most important diseases of wheat. Planting resistant cultivars is effective to control this disease, but resistance can be overcome by new virulence. Races of *Pst* have been characterized by testing isolates on a set of wheat lines with different *Yr* genes, but no avirulence genes have been genetically identified. To identify avirulence genes, we selected 7 *Pst* isolates for re-sequencing and characterized them, together with 7 previously sequenced isolates, for their avirulent/virulent profiles on the 18 *Yr* single-gene differential lines. The 14 isolates had a 1:1 ratio for avirulence and virulence to 12 of the *Yr* genes. From the *Pst* sequences, we characterized 2,146 secreted protein (SP) genes and identified 48,059 high quality variants including 41,764 SNPs and 6,295 Indels in the SP genes. Secretome comparison with other rust fungi identified 923 *Pst*-specific SPs. Effector candidates were identified by characterizing *Pst* SPs based on cysteine-rich, EffectorP predicted, haustorial differentially expressed, *Pst*-specific, and polymorphic among the isolates. Of the 2,091 SPs that fit at least one criterion, 32 fitting all criteria could be considered as effector candidates. We also identified effector candidates significantly associated to avirulence genes corresponding to five *Yr* genes by correlating their polymorphisms to avirulence/virulence profiles. One effector candidate for *AvYr76* was present in all avirulent isolates and absent in all virulent isolates, suggesting that deletion of the gene is a mechanism responsible for changing from avirulence to virulence.

**506T Leptosphaeria maculans effectors involved in the oilseed rape systemic colonization.** *J. Gervais*¹, C. Plissonneau¹, J. Linglin¹, M. Meyer¹, K. Labadie², C. Cruaud², B. Ollivier¹, I. Fudal¹, T. Rouxel¹, M-H. Balesdent¹ 1) INRA, UMR 1290 BIOGER INRA-AgroParisTech, 78850 Thiverval-Grignon, France; 2) CEA-Institut de Génomique, GENOSCOPE, Centre National de Séquençage, Evry Cedex, France.

The stem canker disease, caused by *Leptosphaeria maculans*, is one of the most devastating diseases of oilseed rape (canola). It colonizes the plant in two stages: a short and early colonisation stage corresponding to cotyledon or leaf colonisation, and a late colonisation stage during which the fungus colonises systemically and symptomlessly the plant during several months before stem canker appears. To date, determinants of the late colonisation stage remain poorly understood.

By a transcriptomic approach, we previously identified two waves of effector candidate expression during the early and late colonisation stages (Gervais et al, 2016). The late effector candidates are located in gene-rich genomic regions, whereas the early effector genes are located in gene-poor regions of the genome.

Among the late effector candidates identified, we selected 6 genes for further characterization. We created mutants silenced for these effector candidates. For one of these genes, its expression level correlated negatively with the size of the necrosis observed in the stem.

The identification of new effector genes would contribute to the identification of new resistance genes specific to these effectors. To easily identify matching resistance genes in oilseed rape, we created transgenic isolates expressing these 6 late effectors at the early steps of infection to provide medium-throughput strategies to screen more efficiently different cultivars. Preliminary results indicate that some cultivars with adult resistance were more resistant to these transgenic isolates in cotyledon assays. With this approach, we also identified a cultivar carrying a specific resistance to one these 6 effector candidates.

Reference

Gervais, J., Plissonneau, C., Linglin, J., Meyer, M., Labadie, K., Cruaud, C., Fudal, I., Rouxel, T. and Balesdent, M.H. (2016) Different waves of effector genes with contrasted genomic location are expressed by *Leptosphaeria maculans* during cotyledon and stem colonization of oilseed rape. *Mol. Plant Pathol.*

**507F Delivery of Phytophthora sojae effector Avr1b in planta requires PI3P-binding, but does not require N-terminal cleavage.** *Brett M. Tyler*¹, Qunqing Wang¹, Biao Gu², Felipe Arredondo¹, Eli Perez¹, Shiv D. Kale³ 1) Center for Genome Research and Biocomputing, Oregon State University, ALS3021, Campus Way, OR; 2) College of Plant Protection, College of Plant Protection, Northwest A&F University, Yangling, 712100, China; 3) Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA24061, USA.

A major class of effectors produced by oomycetes contains RxLR motifs that mediate entry of these effectors into plant cells. We previously showed that these effectors can bind to specific lipids including phosphatidylinositol-3-phosphate (PI3P). PI3P-binding requires the RxLR motif, plus in some cases, C-terminal regions of the protein. In order to validate that PI3P-binding mediates host cell entry in planta, we have shown that heterologous PI3P-binding proteins such as yeast VAM7p can functionally replace the RxLR domain of *Phytophthora sojae* effector Avr1b, and can deliver this effector into soybean cells during a natural *P. sojae* infection. The Avr1b and various derivative mutant proteins can be specifically detected in culture supernatants after de-glycosylation, indicating that Avr1b is post-translationally modified. Some RxLR effectors such as Avr1b also undergo N-terminal cleavage following secretion, but others such as Avr4/6 do not. Cleavage of Avr1b occurs upstream of the RxLR motif, and the RxLR motif is not required for cleavage. Mutants of Avr1b
that are not cleaved are delivered normally. We conclude that N-terminal cleavage is not required for delivery in planta. We are now using CRISPR-mediated gene replacement to refine these experiments.

**508W Effectors Required for Epichloë-Wheat Interactions.** Benjamin Moody¹, Christine Voisey¹, Rosie Bradshaw², Paul Maclean¹, Carl Mesarich², Martijn Rep³, Linda Johnson¹ 1) Forage Science, AgResearch Limited, Palmerston North, Manawatu, NZ; 2) Massey University, Palmerston North, New Zealand; 3) Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, The Netherlands.

The fungal endophyte *Epichloë bromicola* forms mutualistic symbioses with grasses of the *Elymus dahuricus* complex. Artificial inoculation studies have shown that *E. bromicola* strains AR3060 and AR3018 are also able to colonise the commercial spring wheat cultivar Monad. Unlike the compatible and symptomless associations they form with their native hosts, colonisation of wheat by AR3060 causes host dwarfing but the endophyte is still able to undergo its life-cycle and be vertically transmitted via seed. AR3018 infection is less compatible and causes severe stunting in wheat, followed by plant death and/or the formation of endophyte-free tillers. Transcriptome analysis of compatible versus incompatible interactions between AR3060 and AR3018 with their native and non-native hosts, show that many genes predicted to encode secreted effector-like proteins are highly up-regulated during interactions with host plants. One of most abundant transcripts in planta encodes a LysM-domain containing protein that has high homology to *Cladosporium fulvum* effector Ecp6 based on its secondary and tertiary structure. Our results indicate that *Epichloë* endophytes may manipulate grass physiology via the production of effector proteins to produce an environment favourable for fungal infection. Through bioinformatics approaches, over 30 *E. bromicola* candidate effectors with potential to regulate compatibility between *Epichloë* fungi and their hosts have been identified. Transient expression of effectors in non-host plants, combined with targeted gene deletion studies, are being conducted to explore their roles in regulating defence responses and symbiotic interactions between *Epichloë* and its novel wheat host. Long term, this study aims to facilitate fully compatible interactions between *E. bromicola* and wheat to enable these fungi to confer biotic and abiotic stress protection to commercial wheat.

**509T Identification of candidate genes encoding the avirulence effector AvrRvi8 from the plant pathogenic fungus *Venturia inaequalis*.** Brogan McGreal¹, Jared Bennett¹, Saskia Bosman¹, Linda Collard¹,³, Cecilia Deng¹, Kim Plummer², Erik Rikkerink¹, Matthew Templeton¹,³, Joanna Bowen¹ 1) The New Zealand Institute for Plant & Food Research, New Zealand; 2) La Trobe University, Australia; 3) The University of Auckland, New Zealand.

The fungus *Venturia inaequalis* causes scab disease of apple. The interaction between the host, *Malus*, and the pathogen follows the 'gene-for-gene' model, with 17 avirulence (*Avr*) and cognate resistance (*R*) gene pairings currently being recognised. Two *R* genes have been cloned from *Malus* (*Riv6* and *Rvi15*), but no avirulence effector genes. Resistance responses in apple governed by 'gene-for-gene' interactions are phenotypically diverse and not restricted to a rapid hypersensitive response. It is hypothesised that the *Rvi8* R protein recognises an effector (*AvrRvi8*), and initiates a stellate necrosis resistance response approximately 10-14 days post inoculation (dpi). Although this response restricts sporulation, rendering the host field-resistant, extensive sub-cuticular fungal growth, typical of a susceptible interaction, is also observed. A bulked segregant/NGS approach was adopted to identify candidate *AvrRvi8* genes. Progeny (80) from an *in vitro* cross between isolates B04 (*AvrRvi8*) and 1639 (*AvrRvi8*) have previously been phenotyped with respect to *AvrRvi8*¹. DNA from two pools (*AvrRvi8* and *avrRvi8*), each comprising 12 progeny, were sequenced using Illumina technology, and 213 candidates for *AvrRvi8*, with SNPs in the orthologues between the pools, identified. Of these, only one candidate was situated between two novel markers for *AvrRvi8* on a single scaffold in the B04 genome. The nucleotide sequence of this candidate gene was identical in the *AvrRvi8* progeny and a further isolate phenotypically classified as *AvrRvi8*, but there were two predicted amino acid substitutions in the sequence from all *avrRvi8* progeny. The candidate gene is predicted to encode a small (200 amino acids with seven cysteines), putatively secreted protein and had a 4 and 6 log2-fold increase in expression in planta compared with *in vitro* at 2 and 7 dpi, respectively, as measured by RNA-seq. The candidate gene belongs to a small gene family and is currently being functionally analysed by a complementation strategy.

¹Broggini et al. (2011) *Fungal Genet Biol.* 48:166-76

**510F A game of omes: hunting for effectors in fungal plant pathogens using comparative genomics.** James Hane¹,², Alison Testa¹, Robert Syme¹, Stefania Bertazonni¹, Chala Turo¹, Mahsa Mousaviderazmahalleh¹,³, Haifei Hu¹, Paula Moolhuijzen¹, Richard Oliver¹ 1) Centre for Crop & Disease Management, Curtin University, Perth, Australia; 2) Curtin Institute for Computation, Curtini niversity, Perth, Australia; 3) The University of Western Australia, Perth, Australia.

The outcome of host-pathogen interactions in plant diseases caused by Fungi is largely determined by the presence or absence of "effector" molecules produced by a pathogen, and corresponding resistance or sensitivity loci in its host. Thus determining the effector component of a pathogen's genome is a crucial step in molecular plant pathology: e.g. in facilitating subsequent isolation of effectors to be used to screen host accessions for resistance/susceptibility loci. However the major fungal taxa abundant in plant pathogens (Pezizomycotina and Basidiomycota) exhibit taxon-specific types of genome mutations and/or a higher observed rate of common types of mutations. Thus, unlike for other microbial pathogens, proteinaceous fungal effectors have proven to be especially difficult to predict, due to an overall lack of sequence conservation. The difficulty in producing a reliable and concise effector candidate list and a limited capacity to experimentally test candidates, has created a strong impetus to develop improved bioinformatic methods for the molecular plant pathology community.

While the plasticity of fungal genomes often obscures clear effector candidates from view, in some cases bioinformatics can turn the tables by leveraging knowledge of these mechanisms and the patterns they leave behind to reveal effectors. We have studied the dynamics of mutation between the genomes of multiple plant-pathogenic fungal species and present methods by which two mechanisms (Repeat-induced point mutation (RIP) and lateral-gene transfer) can be exploited for effector prediction. We have also developed additional methods to support various points in the effector prediction process. These methods and/or the outcomes of prediction across multiple species can be accessed at http://effectordb.com. Additionally our efforts to understand the common and distinguishing properties of fungal effectors have allowed us to make new observations of their defining features, develop new predictive methods and explore parallels with
cytotoxic peptides from a wide range of non-fungal species.

**511W The two-speed genome of Verticillium dahliae mediates emergence of potent virulence factors.** B.P.H.J. Thomma, L. Faino, J. Li, X. Shi-Kunne, J. Depotter, M. Kramer, G.C. van den Berg, D.E. Cook, H. Rovenich, M.F. Seidl Laboratory of Phytopathology, Wageningen University, Wageningen.

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

**512T Investigating pathogenicity and genetic diversity within the clinically relevant Fusarium solani species complex.** L. Wendell, J. Coleman Entomology and Plant Pathology, Auburn University, Auburn, AL.

Members of the genus *Fusarium* are capable of causing severe disseminated infections (fusariosis), which can be difficult to treat and are becoming more common. Most frequently associated with these infections are a number of species within clade 3 of the *Fusarium solani* species complex (FSSC), which are found regularly in the environment. As the FSSC represents >60 phylogenetically distinct species, we were interested in investigating their genetic diversity and variance in pathogenicity. A total of 33 FSSC isolates from clinical sources were compared using multi-locus sequence typing (MLST) and phylogenetic analyses. In accordance with previous studies, all of the isolates collected from patients belonged to clade 3 of the FSSC, and the species most often isolated belonged to *F. keratoplasticum* (FSSC 2), *F. falciforme* (FSSC 3+4), and the currently unnamed species FSSC 5. Karyotypes were analyzed by pulsed-field gel electrophoresis (PFGE) under conditions to evaluate small chromosomes (between 1 and 3 Mb). Every isolate resolved by PFGE was found to have small chromosomes, concentrated between 2.7 Mb and approximately 0.5 Mb. Karyotype diversity was significant between phylogenetic species, and not a single duplicate chromosomal profile was found within any species. All isolates were fitness tested for growth at elevated temperatures, and subsequently screened for virulence in the heterologous host *Galleria mellonella* and a murine model. Those with the greatest ability to grow at 37°C belonged to FSSC groups 2, 3+4, 5, and 20. When tested in animal models, the most virulent isolates were members of FSSC 2, 3+4, and 5. This research demonstrates that phylogenetically closely related species representing a fraction of the FSSC are responsible for a majority of the clinical FSSC infections, and these isolates are both thermotolerant and more virulent than other species within the FSSC.

**513F Functional and molecular characterization of genes involved in antagonisms between two maize endophytes, Fusarium verticillioides and Sarocladium zeae.** M. Gao¹, X. Gu², A. Glenn³, S. Gold³ 1) Department of Plant Pathology, The University of Georgia, Athens, GA; 2) Institute of Bioinformatics, The University of Georgia, Athens, GA; 3) Toxicology and Mycotoxin Research Unit, USDA-ARS, Athens, GA.

*Fusarium verticillioides* (*Fv*) is a prevalent seed-borne maize endophyte capable of causing severe kernel rot and fumonisin mycotoxin contamination. Within maize kernels, *Fv* is primarily confined to the pedicel, while another seed-borne fungal endophyte, *Sarocladium zeae* (*Sz*), is observed in embryos. *In vitro* assay has indicated that *Sz* was able to inhibit the growth of *Fv*. Two lactam-containing antibiotics by *Sz*, named pyrrocidine A (PA) and B (PB), are associated with antagonism of *Fv* by *Sz*. In order to explore the mechanism of antagonism, RNA-seq experiments were conducted by challenging the *Fv* liquid culture with PA and PB at subinhibitory concentrations to induce transcriptomic changes. A total of 27 genes with dramatic induction in both PA and PB treatments, including 17 genes encoding proteins with a beta-lactamase domain, were selected as targets to generate deletion mutants with our OSCAR protocol. Phenotypic and functional analyses of the mutants are being conducted to help us better understand the molecular mechanisms underlying the metabolic antagonism between these fungal endophytes. We will present current results on the bioinformatic analyses of RNA-seq experiments and functional characterization of the related mutants.

**514W StuA is a key regulator of fumonisin production and virulence in Fusarium verticillioides.** Manisha Rath¹, Nicole Crenshaw², Scott GOLD² 1) Department of Plant Pathology, University of Georgia, Athens, GA; 2) Toxicology & Mycotoxin Research, USDA-ARS, Athens, GA.

*Fusarium verticillioides* is one of the most important pathogens of maize, producing fumonisin mycotoxins during infection. Ingestion of fumonisin-contaminated corn causes fatal toxicity in livestock and is associated with neural tube birth defects and growth stunting in children. It is also a potential human carcinogen. It is critical to understand the underlying molecular mechanisms associated with fumonisin production in order to generate novel control approaches. StuA, an APSES class transcription factor, is a global regulator with StuA homologs regulating crucial developmental processes in various fungal species such as sporulation, virulence and toxin synthesis among others. Our objective was to investigate StuA's role in virulence and fumonisin production in *F. verticillioides* via functional and transcriptomic analysis of *?stuA* mutants. The mutants generated via *Agrobacterium*-mediated transformation of *F. verticillioides* with an OSCAR deletion construct, have stunted aerial hyphae and reduced macro- and micro-conidiation. Three of the *?stuA* mutants were selected for transcriptome analysis and cultured in GYAM medium to induce toxin production. A total of 437 genes were differentially regulated across the 3 mutants compared to wild type with ≥ 3-fold change. Out of these, 328 were downregulated and 109 upregulated. Five genes in the FUM (fumonisin) cluster and 8 genes in the FUS (Fusarin C) cluster were dramatically downregulated (≥ 4 fold for FUM and ≥ 8 fold for FUS). Analysis of fumonisin levels in GYAM shows reduced fumonisin production by *?stuA* mutants (≥ 100-fold). Additionally, maize seedlings inoculated with *?stuA* mutants showed greater shoot height and weight as compared to seedlings inoculated with wild type, indicating reduced virulence of the mutants and thus an important role in virulence for the *stuA* gene.
**515T Suppression of genes of Fusarium verticillioides using Virus induced Gene Silencing.** J.E. Jurgenson Biology, University of Northern Iowa, Cedar Falls, IA.

Virus induced Gene Silencing (VIGS) is a technique that can be used to silence genes in plants. Several reports have shown that this approach can also silence genes in organisms (such as pathogens) associated with the plant. Experiments are being conducted that will determine if this technique can be used to silence the genes of the corn pathogen *Fusarium verticillioides* in the same manner that was used to silence stripe rust genes in wheat.(2011, Yin et al.) These experiments involve the introduction of double stranded RNA with sequence homology to selected fungal genes into host corn plants via a virus vector. The ability of subsequent expression of these genes are being evaluated using standard Quantitative PCR techniques. We are using two vectors: one developed from Brome mosaic virus and one from Foxtail mosaic virus.

The objectives of this project are to: 1) Determine the efficacy of *in-planta* expressed RNAs in silencing *Fusarium verticillioides* genes by examining constructs containing sequences derived from genes involved in infection of host corn plants. 2) Use VIGS constructs to conduct high throughput suppression assays of *F. verticillioides* genes to determine if transcripts are essential for fungal pathogenicity or reproduction. *F. verticillioides* genes tested will include those that have homology to other fungal pathogens of corn and to genes of other *Fusarium* species known to be essential for host plant infection. These gene homologs are documented by experimental results published in the literature. 3) Make corn plants that stably express interfering RNAi for genes that show promise for controlling *Fusarium* in VIGS assays.

Chuntao Yin, James E. Jurgenson, and Scot H. Hulbert. (2011) Development of a Host Induced RNAi System in the Wheat Stripe Rust Fungus Puccinia striiformis f. sp. Triticis. MPMI Vol. 24, No. 5, 2011, pp. 554–561

**516F A nonribosomal peptide facilitates cell-to-cell invasion of Fusarium graminearum in wheat.** Leijie Jia, Tinglu Yuan, Dong Zhang, Weihua Tang Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, CN.

*Fusarium graminearum* is an important pathogen of wheat. Deletion of the putative secondary metabolite biosynthetic gene cluster FG3_54 compromised its ability to infect wheat coleoptiles and spikes. Wild-type hyphae invade internal tissues through cell-to-cell penetration, while hyphae of mutants lacking FG3_54 or its member gene FGM4 failed to progress beyond the first layer of wheat cells where papillae-like cell wall depositions were observed. Filtration extracts of FG3_54-expressing *F. graminearum* cultures, when applied to inoculation sites together with FG3_54 deletion strains, facilitated hyphal penetration beyond the first layer of cells. We identified a tripeptide as one of the responsible metabolites. The application of chemically-synthesized tripeptide, restored cell-to-cell invasion in FG3_54-deleted *F. graminearum*, and even enabled colonization of wheat coleoptiles by a banana pathogenic *Fusarium oxysporum* strain. Thus this tripeptide represents an effector that facilitates hyphal cell-to-cell penetration in wheat.

**517W Fusarium G-protein coupled receptors: Modulators of pathogenesis and targets for disease control.** N.A. Brown, T. Dilks, K.E. Hammond-Kosack Plant Biology and Crop Science, Rothamsted Research, Harpenden, Hertfordshire, GB.

*Fusarium graminearum* is a globally important cereal pathogen and mycotoxin producer. When *F. graminearum* lands on a plant, the fungal germling must decide if it is suitable, where to infect, and when to deploy different virulence strategies. But how *F. graminearum* senses the "touch and taste" of its environment through external receptors is largely unknown.

Bioimaging, bioinformatics [1-2] and now transcriptomics has shown how *F. graminearum* hyphae spatiotemporally regulate a complex array of characterised and putative virulence mechanisms during wheat infection. This includes the dramatic up-regulation of genes involved in the biosynthesis of the trichothecene mycotoxin, deoxynivalenol, plus other secondary metabolite clusters, during symptomless infection. Distinct groups of putative secreted effector proteins were up-regulated either during symptomless or symptomatic infection, while hydrolytic CAZyme induction followed a two-step mechanism, resulting in elevated expression during the development of disease symptoms. Collectively, this implies that *F. graminearum* hyphae must sense alterations in the microenvironment within the host and coordinate virulence accordingly.

G-protein coupled receptors (GPCRs) are the largest class of extracellular receptors in eukaryotes and *F. graminearum* possesses more than other model fungi. Interestingly, a group of non-classical GPCRs were highly expressed during the establishment of symptomless infection. Now a collection of *F. graminearum* mutants lacking individual GPCRs genes has been assessed, revealing their involvement in the establishment of wheat infection.

1. Brown N.A. et al (2010) The infection biology of *Fusarium graminearum*: Defining the pathways of spikelet to spikelet colonisation in wheat ears. *Fungal Biology*. 114(7):555-71.

2. Brown N.A. et al (2012) The predicted secretome of the plant pathogenic fungus *Fusarium graminearum*: A refined comparative analysis. *Plos One*. 2012:7(4).

Neil Brown is funded by a Biotechnology and Biological Sciences Research Council (BBSRC) Future Leader Fellowship. Rothamsted Research receives grant-aided support from the BBSRC.

**518T Novel Fusarium graminearum cyclic peptides possess phytotoxic activity.** Linda Harris¹, Adilah Bahadoor², Elizabeth Brauer¹, Whynn Bosnich¹, Danielle Schneiderman¹, Yves Aubin³,⁴, Jeremy Melanson², Izhar Khan¹, Steve Gleddie¹, Barbara Blackwell¹ 1) Ottawa Research & Development Centre, Agriculture & Agrifood Canada, Ottawa, ON, CA; 2) Measurement Science and Standards, National Research Council Canada, Ottawa, ON, Canada; 3) Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, ON, Canada; 4) Department of Chemistry, Carleton University, Ottawa, ON, Canada.

Fusarium Head Blight, a devastating disease of cereal crops in temperate regions globally, is mainly caused by the broad host pathogen *Fusarium graminearum*. A significant number of predicted *F. graminearum* secondary metabolism genes are expressed during host infection but are not yet associated with a metabolite. In an effort to identify and link secondary metabolites to their respective biosynthetic genes, we have isolated two novel cyclic peptides, named gramilin A and B, produced by *F. graminearum* isolates cultured in liquid media.
A combination of LC-HRMS, 1D and 2D-NMR experiments were performed to elucidate their structure. Gene disruption has revealed the nonribosomal peptide synthetase gene responsible for the biosynthesis of these compounds. Gramilin A/B are phytotoxic towards both monocot and dicot plants. Infiltration of either maize or Arabidopsis leaves with gramilin causes cell death within three to four hours. Gramilin A/B do not appear to possess antimicrobial activity against Gram negative or Gram positive human-associated pathogenic bacterial species tested. We are continuing to characterize the mechanism of toxicity.

**519F Transcriptomics and experimental proof that compound appressoria are arsenals of Fusarium graminearum.** M. Mentges¹, A. Glasenapp¹, M. J. Boenisch², A.-L. Martínez Rocha¹, M. Münsterkötter³, U. Güldener³, B. Henrissat⁴, M.-H. Lebrun⁵, J. Bormann¹, W. Schaefer¹ 1) Molecular Phytopathology, Biocenter Klein Flottbek, University of Hamburg, Germany; 2) Cereal Disease Lab, USDA-ARS, St. Paul, United States of America; 3) Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Germany; 4) CNRS & Aix-Marseille Université, Marseille, France ; 5) INRA BIOGER-CPP, Versailles, France.

*Fusarium graminearum* is one of the most destructive plant pathogens. Its infection of cereals causes significant losses due to yield reduction and contamination with mycotoxins, e.g. deoxynivalenol (DON). *F. graminearum* infects the floral leaf by defined structures: runner hyphae are formed for colonization of the floral surface and compound appressoria, e.g. infection cushions, are developed to facilitate multiple penetrations of the plant cell wall.

In this study, we created and compared RNAseq data sets of three different fungal cell types, i.e. infection cushions and runner hyphae (isolated via laser capture microdissection from the surface of paleae), and in culture grown mycelium. Validation of the data by qRT-PCR showed similar gene regulation patterns.

The analysis showed that 3916 genes (about one-third of all genes) are specifically regulated during the infection of the plant compared to mycelium. In total 653 genes are exclusively transcribed: 235 in mycelium, 77 in runner hyphae and 341 in infection cushions.

In particular, we show that 1. in runner hyphae, the highly expressed fungal pigment aurofusarin acts as an antibiotic agent against bacteria and fungi, 2. infection cushions are arsenals of virulence factors, harboring plant cell wall degrading enzymes, specific metabolites like DON, and putative effector proteins, 3. the putative effector protein FgEF1 is localized in the plant cell after colonization by *F. graminearum*.

**520W Ubiquitin-dependent protein degradation during infection structure development by the plant pathogenic fungus Fusarium graminearum.** G. Baermann, W. Schaefer Molecular Phytopathology, Biocenter Klein Flottbek, University of Hamburg, Germany.

The fungal plant pathogen *Fusarium graminearum* is the most important causal agent of Fusarium head blight of small grain cereals and cob rot of maize worldwide. During the initial infection *Fusarium graminearum* forms two morphologically distinct structures, non-invasive runner hyphae and invasive compound appressoria, especially infection cushions.

Transcriptome analyses of runner hyphae and infection cushions showed a high number of genes coding for enzymes involved in the regulation of these structures. In total we identified 158 different fungal genes that are expressed during development on wheat flower leaves and are most likely involved in ubiquitin-dependent protein degradation. Based on sequence homology, there are 19 ubiquitin carboxyl-terminal hydrolases type 1/2, 3 ubiquitin/SUMO-activating enzymes (E1), 23 ubiquitin-conjugating enzymes (E2), 10 ubiquitin-conjugating enzymes with zinc finger motives of the RING-type, 2 Small ubiquitin-related modifier (SUMO) and 53 F-box proteins among these genes.

The project focuses on the role of selected genes during the ubiquitination process in the fungal pathogen, especially during development of fungal infection structures. In a first attempt we deleted five genes which encode for F-box proteins and ubiquitin-conjugating enzymes. Three mutants showed different deficiencies in growth, conidiation, cell wall integrity, ROS stress and virulence.

A detailed analysis of the involvement of protein degradation in the life cycle of *Fusarium graminearum* will be presented.

**521T Fusarium oxysporum and its biocontrol.** Maria-Ermioni Constantin, Francisco de Lamo, Frank Takken, Martijn Rep Molecular Plant Pathology, University of Amsterdam, Amsterdam, North Holland, NL.

*Fusarium oxysporum* (*Fo*) is known as a fungal pathogen capable of causing vascular wilt disease in over 100 different hosts. Although most studies focus on its ability to cause disease, *Fusarium* is also capable of colonizing plants without triggering disease symptoms, as an endophyte. These non-pathogenic strains of *Fo* have been shown to confer protection against Fusarium wilt on tomato caused by *Fo* f.sp. *lycopersici* (*Fo*). We have received from collaborators about 80 *Fo* strains, some of which could have biocontrol potential against wilt disease. These strains were obtained from different locations in Australia, USA and Spain. From the collection, 44 isolates have been selected for genome sequencing. In addition, we developed a high-throughput screening set-up that allows us to test these strains for their ability to confer protection against tomato wilt disease. Preliminary results show variation in the efficiency of protection against wilt disease by non-pathogenic strains. We are currently trying to understand the mechanism behind this protection.

**522F Accessory chromosomes and pathogenicity in Fusarium oxysporum.** JIMING LI, Ido Vlaardingerbroek, Shermineh Shahi, Martijn Rep University of Amsterdam, Amsterdam, NL.

*Fusarium oxysporum* is a species complex, harbouring pathogenic and non-pathogenic strains. Pathogenic strains cause disease in more than 120 different plants, but each pathogenic strain generally causes disease in one or a few related plant species. Comparative genome studies of *Fusarium graminearum*, *F. verticillioides*, *F. solani* and *F. oxysporum* f.sp. *lycopersici* (*Fo*) has shown that *Fo* contains core and accessory chromosomes, and one of the accessory chromosomes contains virulence genes that promote disease in its host tomato. This 'pathogenicity chromosome' can be transferred to a non-pathogenic strain, resulting in acquired pathogenicity. Conversely, loss of the *Fo* pathogenicity chromosome leads to loss of virulence. How does the pathogenicity chromosome transfer happen? Fusion of genetically different strains results in formation of heterokaryons. However, heterokaryons are not stable because they contain different vegetative incompatibility loci, which leads to heterokaryon incompatibility reactions. We have observed that conidial pairing of incompatible *Fo* and *Fo* (non-pathogenic) strains under carbon starvation can result in the formation of viable cells. After formation of heterokaryons, the chromosomes from the *Fo* parental strain are lost, but sometimes one or two chromosomes remain in the progeny, including the
pathogenicity chromosome. We now wish to determine the minimal chromosome regions or genes on the F1 pathogenicity chromosome that can cause disease on tomato plants, and/or can be transferred horizontally. We will also investigate the underlying mechanisms of selective chromosome loss in heterokaryons.

**523W FTF2, the core genome gene of the Fusarium oxysporum FTF family, is involved in conidiation and recognition by the host plant.** Virginia Casado-del Castillo, Jonathan Niño-Sánchez, Francisco J. Hernández-Aparicio, Lizeth Báez-Ojeda, Ernesto P. Benito, José María Díaz-Mínguez CIALE-UNIVERSIDAD DE SALAMANCA, SALAMANCA, SPAIN.

The FTF gene family of *Fusarium oxysporum* encodes Zn(II)₂Cys₆ binuclear cluster DNA-binding transcription factors. We have previously shown that *FTF2* is a single copy gene well conserved in filamentous ascomycetes that is located in the *F. oxysporum* core genome; on the contrary, the *FTF1* paralogues are variable in number, exclusive to *F. oxysporum* and are located in the adaptive genome. Null mutants of *FTF2* in the HV strain FOP-SP1 of *F. oxysporum* f. sp. phaseoli (SP1DFTF2) show reduced virulence when inoculated in common bean plants. The colonization pattern is similar to that displayed by weakly virulent strains (which do not contain *FTF1* paralogues) and the silenced *FTF* mutants, showing a reduction in the number of infected xylem vessels and an increase in parenchymal colonization. However SP1DFTF2 mutants do not show a significative reduction of fungal biomass in colonized plants as it is the case with the *FTF* silenced HV strains. Further functional analysis demonstrated that *FTF2* mutants produce high amounts of macroconida in solid media, but not in liquid media, and induce upregulation of the common bean *PR1* gene which is involved in the plant defensive response mediated by salicylic acid.

To shed light on the specific FTF2-responsive genes, comparative transcriptomic analysis were carried out in a genetic background free of FTF1 paralogues (FOP-SP4/SP4DFTF2). Several genes showing strong differential expression were confirmed by RT-qPCR analysis. Interestingly, the expression of two genes encoding hydrophobin type II proteins was confirmed to be absent in the *FTF2* mutant, and one of them was specifically upregulated during root colonization. These findings indicate that FTF2 is involved in spore production and spore/hypha masking to prevent recognition by the host plant.

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

**524T Deletion of the AP1 transcription factor in the anthropophilic dermatophyte *Trichophyton rubrum* reveals that it is not required for oxidative stress and antifungal drug resistance.** N. T. A. Peres¹,², E. A. S. Lang², V. M. Oliveira², T. A. Bitencourt², A. Rossi², N. M. Martinez-Rossi² 1) Morphology, Federal University of Sergipe, BR; 2) Genetics, Ribeirão Preto Medical School, University of São Paulo, BR.

*Trichophyton rubrum* is the major causative agent of tineas, i.e., skin and nails infections, worldwide. Pathogenesis of tinea is a dynamic and complex process, in which the host inflammatory response and the release of fungal proteolytic enzymes are the major responsible factors for tissue damage during the infection, leading to erythematous and itchy lesions. Functional studies have been helping to understand the mechanisms that allow fungal adaptation to the host environment and stressful conditions. Genetic manipulations are valuable tools to understand the regulation and functionality of genes giving insights into the pathogenesis of fungal infections. However, genetic manipulations of the anthropophilic dermatophyte *T. rubrum* is very laborious given the very low frequency of transformation and homologous recombination. In this work, we describe the obtainment of a null mutant for the AP-1 transcription factor of *T. rubrum*, and its functional characterization in response to several environmental conditions. The knockout strain was obtained by replacing the *ap-1* gene by the *hph* gene that confers resistance to hygromicin B. The gene target cassette was constructed by fusion PCR, and inserted into *T. rubrum* by protoplast transformation. By increasing the size of the flanking homologous regions in the cassette, it was possible to isolate a null mutant strain for the *ap-1* gene. Gene expression analysis revealed that *ap-1* is upregulated during human nail and skin ex vivo infection, growth in keratin, and in response to the exposure to amphotericin B, nystatin, and terbinafine. Functional analyses of the mutant strain revealed that inactivation of the *ap-1* gene did not alter fungal growth or sensitivity to oxidative stress and the antifungal drugs analyzed. However, the null mutant showed a higher growth in nail fragments, hyperpigmentation, and increased conidia sensitivity to calcofluor white, compared to the wild type. These results suggest that in *T. rubrum*, the AP-1 transcription factor is not required for oxidative stress adaptation and antifungal drug resistance, but it is required for fungal growth control during nail infection.

**525F A difference in hydrophobin gene expression profiles among Verticillium isolates corresponds with the gene's highly specific role in resting structure development.** A. Klimes, S. Bibi, T. Q. Nguyen Western New England University, Springfield, MA.

*Verticillium dahliae* causes a vascular wilt disease in a wide range of economically significant crops. The soil-borne pathogen produces melanized resting structures known as microsclerotia that stymie eradication efforts in the field. *Vdh1* is a predicted hydrophobin gene that was among the first *V. dahliae* genes found to be involved in microsclerotia production; in the Race 1 tomato isolate Dvd-T5, disruption of *Vdh1* inhibits microsclerotial development and results in accelerated disease development. We identified a gene that shares 100% nucleotide identity with *Vdh1* in the genome sequence of the Race 2 *V. dahliae* lettuce isolate VdLs.17. Expression of this gene, designated *Vdh1-L*, is down regulated during microsclerotial development. In marked contrast, *Vdh1* was previously shown to be specifically expressed in developing microsclerotia. In *V. alfalfae* (isolate VaMs.102), which produces relatively simple and non-persistent resting structures known as dark resting mycelium, the *Vhd1* homologue *Vdh1-A* shows the expected induction of expression during resting structure development, indicating that repression of *Vdh1-L* during microsclerotia development results from isolate-specific gene regulation patterns. *Vdh1-L*'s role in development appears to be isolate-specific as well, because disruption of *Vdh1-L* did not inhibit microsclerotia production; conversely, under specific nutrient availability conditions, *Vdh1-L* disruption mutants appear to produce more microsclerotia. Our results suggest that developmentally important genes may have highly specific regulation patterns that could be relevant to differences in host-specificity among various *V. dahliae* isolates. Comparison of expression patterns of hypothetical genes during resting structure development in VdLs.17 and VaMs.102 yielded results that support this suggestion: of 9 hypothetical genes whose expression profiles we examined, at least one showed a marked difference in expression between the two species. Comparing genome-scale expression profiles may therefor help us pinpoint important developmental differences between *V. dahliae* isolates with different host-specificities, even in instances of high genome sequence identity.
**526W Functional characterization of Phospholipase D likes in *Phytophthora infestans*.** Harold Meijer¹,², Charikleia Schoina², Shu-tong Wang²,³, Klaas Bouwmeester², Chen-lei Hua², Francine Govers² 1) Wageningen Plant Research, Wageningen University and Research, Wageningen, NL; 2) Laboratory of Phytopathology, Wageningen University and Research, Wageningen, NL; 3) College of Plant Protection, Agricultural University of Hebei, CH.

Invasion of plant host tissue by oomycete pathogens, like *Phytophthora infestans*, is dependent on modifications of the host plasma membrane to facilitate two-way transfer of proteins and other compounds. This modulation of the host plasma membrane, e.g. to establish extrahaustorial membranes, might be accompanied by extracellular (phospho)lipase activities. Phospholipases have been implicated as virulence factors in pathogens and since the *P. infestans* genome harbors a range of small phospholipase Ds (PLD-likes) with a signal peptide, these oomycetes could use similar weaponry during the infection process. We show that three small PLD-likes, belonging to three PLD classes, are expressed during various oomycete life-stages, including infection. *In planta* expression of small PLD-likes promotes local cell-death which highly depends on silencing suppression, calcium presence and intact catalytic motifs. We further demonstrate that *in planta* expression of the wild type small PLD-likes facilitates *P. infestans* infection and supports sporangia formation. These results suggest that the small PLD-likes are catalytically active and function by executing membrane modifications to support *Phytophthora* growth and development *in planta*.

**527T *CgXbp1* modulates temporal transcriptional dynamics in human fungal pathogen *Candida glabrata* during interaction with host macrophages.** M. N. Rai, C. Parsania, R. Rai, K. H. Wong Faculty of Health Sciences, University of Macau, Macau, MO.

*Candida glabrata*, an opportunistic human fungal pathogen in immunocompromised patients, remodels its carbon metabolism to survive in nutrient-deprived intracellular macrophage microenvironment. Here, we have identified a transcription factor, *CgXbp1*, required for remodeling carbon metabolic pathways in *C. glabrata* upon exposure to host macrophages. Employing the next generation sequencing approaches, we showed that *Cgxbp1Δ* mutant exhibited constitutively active fatty acid catabolic and proteolytic pathways even in the presence of sufficient carbohydrate in the growth medium, suggesting that carbon metabolic processes are deregulated upon *CgXBP1* deletion. Next, we demonstrated that *CgXbp1* is essential for arresting the cells in G1 phase of cell cycle during carbon starvation. Global gene expression analysis on *Cgxbp1Δ* mutant revealed that genes associated with GO terms – mitochondrion organization, mitochondrial transport and mitochondrial translation were highly enriched among down-regulated genes, indicating that *CgXbp1* may have pivotal roles in cellular energy homeostasis maintenance. Comparison of the transcriptional profiles of macrophage-engulfed wild-type and *Cgxbp1Δ* mutant cells revealed that unlike wild-type cells, *Cgxbp1Δ* mutant was unable to repress genes associated with GO terms - Translation, Ribosome biogenesis and RNA metabolic processes during early stage of macrophage infection. Overall, our gene expression data indicated that *CgXbp1* not only is central in carbon metabolic remodeling, but also affects global transcriptional response of *C. glabrata* cells in host macrophages.

**528F Transcriptome changes in a seed-transmitted endophyte during host floral development are related to stress and reactive oxygen responses.** Padmaja Nagabhyru¹, Randy D. Dinkins², Christopher L. Schardl¹ 1) Plant Pathology, University of Kentucky, Lexington, Kentucky, KY; 2) Forage-Animal Production Research Unit, USDA-ARS, Lexington, Kentucky, KY.

*Epichloë coenophiala* is the common systemic symbiont (endophyte) of tall fescue (*Lolium arundinaceum*), the most widely planted cool-season forage grass in the U.S., and the fungal endophyte strictly and efficiently transmits vertically by colonizing floral primordia, ovaries and embryos. We investigated differences in endophyte and host transcriptomes associated with the plant reproductive tissues, as well as vegetative pseudostems and floral bracts. Remarkably, ovaries of *Epichloë*-symbiotic plants showed almost no significant differences in gene expression compared to those of asymbiotic plants. In contrast, 277 fungal genes showed significantly higher expression in ovaries than pseudostems, and many of these were indicative of responses to stress and reactive oxygen species (ROS). Examples are genes for heat-shock proteins, trehalose biosynthesis enzymes, catalases, superoxide dismutases, and bax inhibitor-1 protein. Gene expression profiles of floral primordia and bracts indicated that this endophyte response began early in host floral development. Out of 150 fungal genes with significantly higher expression in pseudostems compared to ovaries, many were alkaloid biosynthesis genes, or sugar and amino acid transporter genes. The major changes in the fungal transcriptome during plant reproductive development indicate a shift away from specialized metabolism and towards enhanced stress tolerance, and suggest that response to ROS is important for vertical transmission of the endophyte.

**529W RNA-sequencing of susceptible and resistant spinach downy mildew interactions.** Steven J. Klosterman¹, Roberto Ornelas², Kevin Stoffel³, Amanda Hulse-Kemp³, Sebastian Reyes-Chin-Wo³, Lida Derevnina³, Steven T. Koike⁴, Richard Michelmore³, Allen Van Deynze³ 1) USDA-ARS, Salinas, CA; 2) California State University, Monterey Bay, Seaside, CA; 3) University of California, Davis, Davis, CA; 4) University of California, Cooperative Extension, Salinas, CA.

*Peronospora effusa* is an oomycete plant pathogen that causes downy mildew, a widespread and destructive disease on spinach. Symptomatic spinach leaves infected with *P. effusa* display yellow lesions in an otherwise dark green background, severely limiting their fresh market value. In this study, RNA sequencing was performed to detect gene expression in the pathogen and host in *P. effusa*-spinach interactions. Susceptible and resistant spinach cultivars, Viroflay and Solomon, respectively, were inoculated with *P. effusa*, while control plants of both cultivars were uninoculated. As both host and pathogen genome resources are available, gene expression levels of *P. effusa* and the two inoculated cultivars were evaluated by RNA-sequencing. We detected over 700 different transcripts of the pathogen as expressed in the inoculated Viroflay leaves, including two genes encoding effector homologs. The results of this work provide initial insights into genetic responses of *P. effusa* during infection, and also the resistant and susceptible host responses.

**530T Identification and Prevalence of Proteins Predicted to be Intrinsically Disordered in the Secretome of the Fungal Plant Pathogen *Venturia inaequalis*.** L. Collard¹,², C. Deng¹, M. Templeton¹,², E. Rikkerink¹, K. Plummer³, J. Bowen¹ 1) The New Zealand Institute For Plant & Food Research, Auckland, NZ; 2) The University of Auckland, Auckland, New Zealand; 3) La Trobe University, Melbourne, Australia.

Apple scab disease is caused by the Ascomycete fungus, *Venturia inaequalis*, an apoplastic dwelling and host-specific pathogen. This pathosystem is consistent with the gene-for-gene model of resistance interactions and 17 avirulence (*Avr* gene) and resistance (*R* gene)
interactions have been identified thus far. As yet, no effectors involved in the suppression or evasion of host defence have been characterised in this organism.

Several hundred candidate effector genes were identified in draft whole genome sequences (WGS) of a set of five isolates of *V. inaequalis* with differential virulence phenotypes against hosts containing specific resistances (MNH120, B04, 1639, Race 5 on *Malus* × *domestica* cultivars and 1389 on *Eryobotria japonica*). Criteria for effector prediction included: low molecular weight (less than 500aa), secretory signals, differentially upregulated expression during infection compared with *in vitro* expression, cysteine enrichment and lack of sequence similarity to known proteins. This large candidate gene set was further bioinformatically interrogated to prioritise a subset for functional characterisation. Disulphide bridges are important structural elements thought to confer stability of proteins in the protease rich apoplast and various configurations of cysteine bridges were predicted in candidates using sequence based prediction tools. Highly stable configurations including inhibitory cysteine knots (ICKs) were detected in some candidates. In addition to these elements, regions of intrinsic structural disorder were frequently detected in the candidates. Lack of secondary structure could enable manipulation of protein-protein interactions, diversity of function of a protein, sensitivity to host cues or evasion of recognition by host resistance proteins. The sensitivity of many effector prediction tools may be enhanced by inclusion of IDP consideration. Candidate atg7917, in the *V. inaequalis* isolate MHN120, contains a relatively large disordered segment with an ICK at the C terminal and has been prioritised for functional analysis using knock out and protein expression strategies.

**531F Characterization of the interaction between bacteria and Magnaporthe oryzae.** X. Zhang, JR Xu Botany and plant pathology, Purdue University, west Lafayette, IN.

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

**532W Application of Host-Induced Gene Silencing (HIGS) for The Control of Rice Blast.** M. Wang, R. Dean Department of Entomology and Plant Pathology, NCSU, Raleigh, NC.

Rice blast, caused by the hemibiotrophic fungus *Magnaporthe oryzae*, is the most destructive disease of rice world-wide. To overcome readily broken-down disease resistance, HIGS (host induced gene silencing) is being developed as a strategy to create a new type of disease resistant plants. Eight genes (*CRZ1*, *Pmc1*, *MagB*, *Lhs1*, *CYP51A*, *CYP51B*, *Exo70*, *Sec5*) that play important roles for the pathogenicity and development of *M. oryzae* were chosen to make HIGS vectors. The vectors were transformed into rice calli through the *Agrobacterium*-mediated method, generating RNA silencing signals in transgenic rice that will silence the target genes of invasive fungus. T0 and T1 generations of transgenic rice plants were generated and the insertion of HIGS fragment confirmed by genomic DNA PCR and Southern blotting. Generation of siRNAs was confirmed by Northern blotting. Silenced and control transgenic rice plants have been challenged with *M. oryzae* and the results will be presented. In addition, treating *M. oryzae* with naked diced siRNA derived from the target genes silenced their expression in the fungus and caused functional collapse. These findings suggest RNA silencing signals can be transferred from host to invasive fungus and HIGS has potential to generate resistant rice against *M. oryzae*.

**533T Functional characterization of SUMOylation-associated genes in the rice blast fungus, Magnaporthe oryzae.** You-Jin Lim¹,
Yong-Hwan Lee¹,² 1) Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Korea; 2) Center for Fungal Genetic Resources, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea.

Among the post-translational modifications, SUMOylation is widely conserved process of attaching SUMO (small ubiquitin related modifier) to a substrate protein in eukaryotic organisms. This process controls many important biological mechanisms including transcriptional regulation, protein localization, cell cycling, DNA repairing, and pathogenesis. In *Saccharomyces cerevisiae*, SUMOylation is required for DNA repair, growth, sporulation, and stress resistance. However, SUMOylation is still uncharacterized in plant pathogenic fungi including *Magnaporthe oryzae*. The SUMO-activating enzymes (E1), SUMO-conjugating enzymes (E2), SUMO ligases (E3), and SUMO protease are the four main enzymes in the SUMOylation. In this study, to elucidate the roles of SUMOylation in fungal development and pathogenicity, four SUMOylation-associated genes encoding two E1 (*MoAOS1* and *MoUBA2*), one E2 (*MoUBC9*), and one SUMO protein gene (*MoSMT3*) were functionally characterized by gene deletion strategy. All deletion mutants of four genes, Δ*Moaos1*, Δ*Mouba2*, Δ*Moubc9* and Δ*Mosmt3*, showed significant defects on mycelial growth, conidiation, conidial morphology, conidial germination, appressorium formation, and pathogenicity. These data suggest that SUMOylation is essential for fungal development and pathogenicity in *M. oryzae*. This study would enhance to comprehensively understand the role of the SUMOylation in the rice blast fungus and beyond.

**534F Genome-wide profiling of small RNA transcriptome at single nucleotide level in the rice blast fungus.** G. Choi¹, J. Jeon¹,
Y.H. Lee¹,² 1) Interdisciplinary Program in Agricultural Genomics, Seoul National University, Seoul 08826, Korea; 2) Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea.

RNA silencing is the universal strategy to regulate diverse biological pathways. Since RNA-induced silencing complex inhibits complementary mRNA activity, sequence changes by single nucleotide polymorphism (SNP) or RNA modification may affect the target process. In fungi, post-transcriptional gene silencing is known to be involved in genome stability and viral defense. Functional small RNAs
were also predicted in several fungal species including plant pathogens. However, the role of the fungal small RNAs was not well understood in the pathogen-host interactions. As a first step to decipher the roles of small RNAs in the rice blast fungus, *Magnaporthe oryzae*, small RNA transcriptome data from in planta and under stress conditions were collected and analyzed. Small RNA reads were matched to *M. oryzae* genome and read counts were normalized using upper-quartile scaling. Sequences were classified as specific and conserved based on the number of libraries where they were expressed. Higher abundance and lower diversity were detected in conserved sequences compared to specific sequences. Contrary to conserved sequences, specific sequences were mapped more on the genic region than the repeat region across libraries. Small RNA reads matched to the genome with one mismatch were also analyzed compared to perfectly mapped reads. Mismatches were enriched at the 3' end position of these reads, which may imply post-transcriptional modification of small RNAs. Some imperfectly matched small RNAs had the higher copy numbers than perfectly matched small RNAs on the corresponding chromosomal position. These data suggest that those sequences often discarded in the small RNA analysis pipeline could be functional as isomers. This study would provide new insights on understanding the roles of fungal small RNAs in the rice blast fungus and beyond.

**535W Structure of a fungal heme-iron acquisition pathway.** Ziva Weissman¹, Shir Yaish¹, Lena Nasser¹, Udita Roy¹, Cristina Avila¹, Guy Horev², Hay Dvir³, *Daniel Kornitzer*¹ 1) Faculty of Medicine, Technion - I.I.T., Haifa, Israel; 2) Bioinformatics Knowledge Unit, Technion - I.I.T., Haifa, Israel; 3) Technion Center for Structural Biology, Technion - I.I.T., Haifa, Israel.

Iron acquisition poses a general challenge for all organisms due to the low solubility of elemental iron, and a particular challenge for pathogenic microorganisms due to the iron withholding mechanisms deployed by the host. Many microbial pathogens have therefore evolved mechanisms for extracting iron from hemoglobin, the largest iron store in the host. In fungi such as *Candida albicans*, the heme-iron utilization pathways includes an extracellular cascade of small secreted and GPI-anchored proteins containing a CFEM domain, defined by a sequence signature consisting of 8 cysteines. These proteins all are able to capture heme from hemoglobin and to exchange heme among themselves, consistent with a pathway in which heme is extracted from hemoglobin outside the cell and is then transferred from one CFEM protein to the next across the cell wall, until it reaches the plasma membrane. We solved the structure of the hemophore Csa2, and found that its CFEM domain adopts a novel protein fold, held together by 4 disulfide bonds generated by the 8 conserved cysteines. Heme is bound via a flexible N-terminal extension and a unique redox-sensitive heme-iron coordination mechanism, consistent with rapid ligand binding and release. At the plasma membrane, the heme is endocytosed by a mechanism involving the ESCRT pathway and a β-arrestin. To identify the predicted transmembrane heme receptor that connects the extracellular CFEM network with the endocytic pathway, we turned to phylogenetic profiling: taking advantage of the hundreds of fungal genomes available, we screened for genes jointly present across fungi with the CFEM heme transfer cascade genes. This enabled us to identify a new class of plasma membrane proteins that is essential for heme uptake. The hemoglobin-iron utilization pathway that emerges from these studies, which is distinct from bacterial systems, indicates that fungi have evolved a unique solution to the heme-iron acquisition problem. Phylogenetic profiling is shown here to represent a powerful method for unraveling cellular pathways in organisms that are recalcitrant to conventional genetic screening.

**536T Genome wide expression profiling of the response of Neurospora crassa to the antifungal plant defensin MtDef4.** Z.Allen. *Snodgrass*¹, K. El Mounadi¹, D. Shah² 1) Biology, Kutztown University of Pennsylvania, Kutztown, PA; 2) Donald Danforth Plant Science Center.

Defensins are small, cysteine-rich antimicrobial proteins present in all plants. MtDef4 from *Medicago truncatula* is an evolutionarily conserved plant defensin that exhibits strong antifungal activity towards an array of fungal pathogens. Overexpression of this defensin in transgenic plants significantly increases the resistance of these plants to fungal pathogens. In order to understand the mechanisms by which MtDef4 mediates its antifungal activity, we have investigated the changes in the gene expression profile of *Neurospora crassa* in presence of the defensin. A total of 254 genes were differentially expressed in response to MtDef4 and encoded proteins involved in various metabolic and cellular processes. The majority of genes highly expressed in presence of the defensin control cellular stress responses. Other up-regulated genes are involved in DNA and protein synthesis, cell cycle control, signal transduction, regulation of gene expression and lipid metabolism. Deletion mutants of several up-regulated genes were further characterized to determine the contributions of these genes to MtDef4's mode of antifungal action.

**537F Molecular and cellular responses of the ascomycete fungi Neurospora crassa and Fusarium graminearum to the antifungal plant defensin MtDef4.** K. El Mounadi¹, K. Islam², D. Shah² 1) Biology, Kutztown University of Pennsylvania, Kutztown, PA; 2) Donald Danforth Plant Science Center.

Defensins play an important role in plant defense against fungal pathogens. The plant defensin, MtDef4, inhibits the growth of the ascomycete fungi, *Neurospora crassa* and *Fusarium graminearum*, at micromolar concentrations. Previous studies have suggested that MtDef4 is transported into the cytoplasm of these fungi and exerts its antifungal activity on various intracellular targets. Here, we show that *N. crassa* and *F. graminearum* respond very differently to MtDef4 challenge. MtDef4 permeabilizes the plasma membrane of *F. graminearum*, but not of *N. crassa*. We find that it is targeted to different subcellular compartments in each fungus. Internalization of MtDef4 in *N. crassa* is energy-dependent and involves endocytosis. By contrast, MtDef4 appears to translocate into *F. graminearum* autonomously using partially energy-dependent pathway. MtDef4 has been previously shown to bind to the phospholipid phosphatidic acid (PA). We provide evidence that the plasma membrane localized phospholipase D (PLD1), involved in the biosynthesis of PA, is needed for entry of this defensin in *N. crassa*, but not in *F. graminearum*. To our knowledge, this is the first example of a plant defensin which inhibits the growth of two ascomycete fungi via different mechanisms.

**538W Perturbation of Tim54 expression affects Hyphal Interference and increases life-span in Podospora anserina.** Alex Mercier, Colin Clairet, David Morais, Philippe Silar, *Sylvain Brun* LIED-UMR 8236, Univ Paris-Diderot, Paris, FR.

Fungi are very successful microorganisms capable of colonizing virtually any ecological niche where they must constantly cope with competitors such as other fungi, bacteria, nematodes, etc. We have shown that the ascomycetes *Podopora anserina* exhibits Hyphal Interference (HI), an antagonistic response triggered by direct contact of competing fungi hyphae (Ikediugwu and Webster, 1970). Indeed, when challenged by *Penicillium chrysogenum*, *P. anserina* produces hydrogen peroxide at the confrontation and kills the hyphae of *P*.
*chrysogenum* (Silar, 2005). This analogy with the oxidative burst taking place during innate immune response in higher eukaryotes was further strengthened when we demonstrated that as in animals and in plants, HI requires the NADPH oxidase (Nox) PaNox1 in *P. anserina* (Silar, 2005).

Here, we report the characterization of the *PDC*<sup>2218</sup> mutant affected in HI. When challenged with *P. chrysogenum*, the *PDC*<sup>2218</sup> mutant produces a massive oxidative burst at the confrontation compared to the wild type but this increased production of hydrogen peroxide is not correlated to a higher mortality of the cells of *P. chrysogenum*. Hence, the oxidative burst and cell death of the challenger are uncoupled in *PDC*<sup>2218</sup>. We first proceeded to the positional cloning of the mutation and identified a deletion spanning 1560 bp in the mutant genome. Even though the deletion disrupts the *PaNip30* putative CDS as well as the 3' UTR (UnTranslated Region) of the neighboring *PaCdk5* putative gene, we eventually show that neither *PaCdk5* nor *PaNip30* play a role in HI. Instead, the gene responsible for the phenotype of the *PDC*<sup>2218</sup> mutant is located more than 1000 bp away from the deletion. This gene is *PaTim54* encoding the homologue of the budding yeast mitochondrial internal membrane import machinery component Tim54p. We show that *PaTim54* is essential in *P. anserina* and that the phenotypes displayed by the hypomorphic *PDC*<sup>2218</sup> mutant (renamed *PaTim54*<sup>2218</sup>) is the consequence of a drastic reduction in the expression of *PaTim54*. Finally, the pleotropic *PaTim54*<sup>2218</sup> mutant displays increased life-span, a phenotype in accordance with its observed altered mitochondrial physiology.

Ikediugwu, F.E., and Webster, J. (1970). Antagonism between Coprinus heptemerus and other coprophilous fungi. MYRETR Trans. Br. Mycol. Soc. **54**, 181,1N2-204,1N4.

Silar, P. (2005). Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant. Mycol. Res. **109**, 137–149.

**539T Characterization of necrosis-inducing NLP proteins in *Leptosphaeria maculans* and *L. biglobosa*.** Georgia Mitrousia¹,
Pryank Patel¹, Henrik Stotz¹, Henk-Jan Schoonbeek², Christopher Ridout² 1) School of Life and Medical Sciences, University of Hertfordshire, Hatfield, AL10 9AB, UK; 2) Department of Crop Genetics, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

Proteins of the superfamily of necrosis and ethylene-inducing peptide 1 (Nep1) like proteins (NLPs) are produced by various phytopathogenic microorganisms. These secreted proteins have been studied in bacteria, fungi and oomycetes. They are involved in cell death but also in activation of host innate immune responses (Qutob et al. 2006). Protein prediction following the *Leptosphaeria maculans* genome sequencing has identified two similar-to-Nep1 proteins (Lm_NLP1 and Lm_NLP2). Recent work showed that Lm_NLP1 is required for the complete virulence of *L. maculans* (Haddadi et al. 2015). Here, the Lb_NLP gene was identified in the *L. biglobosa brassicae* genome, based on the conserved GHRHDWE heptapeptide motif that is characteristic for the NLP family. The three NLPs from the *Leptosphaeria* spp. were found to belong in the Type I of NLPs, based on their amino acid sequence (Gijzen and Nürnberger 2006) in a conserved region. We observed that a peptide motif derived from a fragment of type I NLPs from bacteria, oomycetes and fungi that is not required for necrosis can induce PAMP triggered immunity in *Arabidopsis*. Further, a 24 amino acid peptide from Lm_NLP1 is recognised by some *Arabidopsis* and *Brassica* lines as a PAMP and induces a transient ROS-burst. Recombinant proteins of the three identified *Leptosphaeria* NLPs with or without the signal peptide were used to examine their cytotoxic activity. This will enable us to determine how the different proteins exert their biological activities. Analysis of RNAseq data of *B. napus* seedlings following *L. maculans* inoculation revealed the up-regulation of four NEP-1 interacting genes. The role of these genes following infiltration of the recombinant proteins is discussed.

**References:**

Qutob et al. 2006. The Plant Cell, **18**, 3721–3744.

Haddadi et al. 2015. Molecular Plant Pathology **17**, 1196-1210.

Gijzen and Nürnberger 2006. Phytochemistry **67**, 1800–1807.

**540F Brachypodium distachyon - A new model host for *Claviceps purpurea*.** S. Kind, S. Schurack, J. Hinsch, P.

Tudzynski University of Muenster, Muenster, DE.

The ergot fungus *Claviceps purpurea* is a biotrophic phytopathogen with a broad host range including important crop species. The infection is highly specific to unfertilized ovaries and causes intoxication of the harvest with ergot alkaloids, thus, it is unsuitable for food and feed (Hinsch and Tudzynski, 2015). To study the infection process rye is currently used as standard host but its cultivation conditions are complex, its large genome hinders genetic modifications and generally there is only little knowledge about typical pathogen responses. In contrast, the grass species *Brachypodium distachyon* combines several qualities of a suitable model host and allows new perspectives for molecular analysis of the infection process (Fitzgerald et al 2015). Its susceptibility to *C. purpurea*- infection was tested by the appearance of macroscopic infection symptoms, microscopic observation and altered expression profiles of typical disease responsive genes. Our results show that *B. distachyon* and *C. purpurea* can establish a compatible interaction featuring micro- and macroscopic infection symptoms. However, the appearance of macroscopic symptoms is reduced compared to rye which hinders statistically significant evaluation. We explored different methods to quantify the infection process such as determination of the ratio from fungal to plant biomass and plant gene expression. We observed that plant responses matches results from known compatible interactions. Taken together, those tools enable us to study the interaction between *C. purpurea* and Brachypodium. We want to use this system to test the general relevance of virulence factors from the *Claviceps*-rye system and compare the plant response to *C. purpurea* to other pathogens with regard for fungal derived phytohormones, especially cytokinins.

Hinsch and Tudzynski in Patterson and Lima (eds) Mol. Biol. of [...] Mycotoxigenic and Mycotic Fungi (2015): 229

Fitzgerald et al. "Brachypodium as an emerging model for cereal-pathogen interactions." Annals of botany (2015): 717

**541W Identification of host targets for effectors from the anther smut fungus, *Microbotryum lychnidis-dioicae*.** V.S kuppireddy,
M.C Tsai, C Cahill, B Carman, M.H Perlin Department of Biology, University of Louisville, Louisville, KY.

The anther smut disease caused by *Microbotryum lychnidis-dioicae* and its close relatives may be considered a sexually transmitted disease, spreading from host-to-host via pollinators that would normally transmit the means of host reproduction. In the male plant, the fungus replaces the pollen with its own spores, whereas in female plants the ovaries abort and develop spore bearing pseudoanthers. Fungal pathogens have evolved highly advanced mechanisms to engage their hosts in intimate contact and sabotage host immune
responses; commonly, this is done by secreting effector proteins into host cells to target regulators of defense or plant development. Many of these small secreted proteins, specifically produced during plant infection, manipulate host cells to facilitate parasitic colonization by biotrophic fungi, e.g., by suppressing plant innate immunity or enhancing nutrient availability. However, the roles of these predicted effectors and how they perform their functions remain unresolved. We have characterized the roles of several effectors by identifying their host targets using the yeast 2-hybrid technique to identify protein-protein interactions. Some interesting potential plant proteins that will interact with these fungal effectors were identified, including at least one predicted to be involved in pollen tube elongation. Further experiments will reveal the significance of these fungal effectors in pathogenicity and also their possible location inside the host plant once it is infected. This overall study will provide more insights into the mode of pathogenicity of this fungus and explore not just how a biotroph behaves inside a host but also reveal aspects of the symbiotic behavior between the host and the fungus.

**542T Secreted Nep1-like proteins of oomycetes, fungi, and bacteria trigger immunity in Arabidopsis; genetic dissection of NLP-induced plant defense.** Tom Raaymakers, Ruben Hijne, Tijmen Van Butselaar, Guido Van den Ackerveken Plant-Microbe Interactions, Utrecht University, Utrecht, NL.

Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by a wide range of plant-associated microorganisms, in particular by many phytopathogenic oomycetes and fungi. Both cytotoxic and non-cytotoxic NLPs act as a microbe-associated molecular pattern (MAMP) in *Arabidopsis thaliana*. Specifically, a 24 amino acid fragment, nlp24, derived from a conserved region of oomycete, fungal, and bacterial type 1 NLPs triggers defense. The widespread distribution of NLPs makes this family the first proteinaceous MAMP identified in three different kingdoms of life. A receptor complex consisting of RLP23-SOBIR1-BAK1 mediates NLP-triggered immunity (NTI) in *Arabidopsis*. Here, we present our data on the genetic dissection of NTI, using a transgenic *Arabidopsis* line expressing an estradiol-inducible version of *HaNLP3* of the Arabidopsis downy mildew pathogen *Hyaloperonospora arabidopsidis*. When treated with estradiol plants become severely stunted and plant immunity is strongly activated. M2 seeds of an EMS-mutagenized population were screened for loss of NLP-triggered growth inhibition. We selected ~40 mutants showing normal growth after estradiol treatment and insensitivity to the nlp24 peptide. The obtained *decreased NTI (dni)* mutants are analysed for defects in responsiveness to other MAMPs and their susceptibility to pathogen infection. We will report on our progress on *dni* mutant analysis and cloning of the corresponding *DNI* genes.

**543F Taking a host genetics approach to understand how the intracellular fungal pathogen Histoplasma capsulatum survives within and lyses host macrophages.** A.L. Cohen¹, N.V. Prooyen¹, M.C. Bassik², A. Sil¹ 1) Microbiology and Immunology, UCSF, San Francisco, CA; 2) Department of Genetics, Stanford University School of Medicine, Stanford, CA.

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

**544W MicroRNA expression during Metarhizium anisopliae-host pathogenic interaction.** M.A.K. Alenezi¹, M. Hitchings², D.C. Eastwood¹ 1) Department of Biosciences, Swansea University, Swansea. GB; 2) School of Medicine, Swansea University, Swansea. GB.

Entomopathogenic fungi, such as *Metarhizium* spp., target a wide range of insect species and have been employed as biological control agents reducing the need of chemical pesticides. Infection of an insect host involves a developmental progression from spore attachment and germination, host cuticle degradation and penetration, systemic proliferation as blastospores, and late stage mycelial growth and sporulation. Pathogenic determinants and gene expression during the infection process have been studied, while mechanisms regulating morphogenetic change are still to be fully elucidated. This study aimed to determine whether microRNAs have a functional role during the infection time course of *Galleria mellonella* (wax moth) larvae with *Metarhizium anisopliae*. Methodology for miRNA extraction and was developed and, following Illumina MiSeq sequencing, *Metarhizium*-specific miRNAs differentially expressed at different stages of infection that align to coding and non-coding regions of the *M. robertsii* genome sequence were identified.

**545T Alteration of polyamine metabolism negatively affects Aspergillus flavus development and pathogenesis during its interaction with corn kernels.** R. Majumdar¹, J. Cary¹, S. Minocha², M. Lebar¹, B. Mack¹, C. Sickler¹, C. Carter-Wientjes¹, K. Rajasekaran¹, D. Bhatnagar¹ 1) Food and Feed Safety Research Unit, USDA-ARS, New Orleans, LA; 2) Dept. of Biological Sciences, University of New Hampshire, Durham, NH.

Polyamines (PAs) are low molecular weight polycations that regulate DNA replication, transcription and translation in living cells. They have been shown to positively influence growth, development, and pathogenicity in both plant and human pathogens. Among the three predominant polyamines (putrescine, Put; spermidine, Spd; spermine, Spm), Spd is absolutely required for cell division. Spermidine (triamine) is synthesized from Put (diamine) by spermidine synthase. Spermidine serves as the donor of a 4-aminobutyl group to the Lys50 residue of eukaryotic elongation factor 5A (eIF5A) to produce the rare amino acid, hypusine. Hypusination-mediated activation of eIF5A is critical for initiation of translation. *A. flavus* is an opportunistic fungus that infects corn and other oilseed crops and produces the toxic and carcinogenic mycotoxins, aflatoxins, upon infection. To explore the role of spermidine synthase (*spds*) in *A. flavus* pathogenesis, we disrupted the single copy of the *spds* gene and studied the ability of *spds* mutants to grow on artificial media as well as infected corn
kernels. Inactivation of *spds* significantly reduced mycelial growth and sporulation on artificial media and an exogenous supply of Spd (auxotroph) was required to support fungal growth and development. Infection of corn kernels using *spds* mutant, shows significantly less fungal growth and sporulation (vs. controls). These results suggest that impairment of Spd biosynthesis negatively affects *A. flavus* pathogenesis and pre-treatment (prior to seed inoculation) of a *spds* mutant with Spd and possible Spd acquisition from the host, are not sufficient to restore wild-type levels of pathogenesis during seed infection. On the other hand, dual disruption of the ornithine decarboxylase (*odc*) gene (involved in Put biosynthesis) and a putative PA uptake transporter had little effect on fungal growth and sporulation. The significance of these observations will be discussed in context to the expression of *A. flavus* genes associated with polyamine metabolism (biosynthesis, uptake, and catabolism) and aflatoxin biosynthesis, and aflatoxin contents in the *spds* mutant vs. wild-type *A. flavus* infected corn kernels.

**546F Ramularia collo-cygni and barley host-pathogen interactions.** *E. Sjökvist*¹,⁵, R. Lemcke², J.-B. Lopez³, M. Kamble³, S. Visentin³,⁴, M. Blaxter⁵, A. Newton⁴, E. James⁴, S. Dam³, R. Hjortshøj⁶, N. Havis¹, M. Foged Lyngkjær², S. Radutoiu³ 1) SRUC, Crop and Soil, Edinburgh, UK; 2) Department of Plant and Environmental Sciences, University of Copenhagen, Denmark; 3) Department of Molecular Biology and Genetics, Aarhus University, Denmark; 4) The James Hutton Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK; 5) Institute of Evolutionary, Biology School of Biological Sciences, University of Edinburgh, UK; 6) Sejet Planteforædling, Horsens, DK.

Ramularia collo-cygni is a fungal pathogen of barley, and the causal agent of Ramularia leaf spot (RLS), which is now a major economic disease in many countries. R. collo-cygni has a global distribution and is spread both via the barley seed and through airborne conidiospores. The fungus has a long asymptomatic growth stage in the host plant and only causing necrotic spots after the plant experiences a stress e.g. waterlogging or flowering. To uncover the underlying genetics of the R. collo-cygni host/pathogen interaction and the transition from an endophytic to necrotrophic lifestyle we have combined RNA-sequencing, genome sequencing, proteomics, effector prediction and microscopy work. We will present the up-to-date results from this on-going Danish government funded collaborative project.

**547W VdNUC-2 regulates *Verticillium dahliae* infection through a novel Signaling pathway.** *S. Deng*, C.Y. Wang, S.Q. Luo, X. Zhang, Q. Wang, L. Lin Jiangsu Academy of Agricultural Sciences, Nanjing, China.

**Purpose:** In soil-borne fungal vascular wilt pathogen *Verticillium dahliae*, we characterized a pathogenicity-related gene *VdNUC-2*, named after its homolog *NUC-2* in *Neurospora crassa*. *NUC-2* and its homologs (such as *Pho81* in *Saccharomyces cerevisiae*) belong to cyclin-dependent kinase inhibitor, and are known for their essential roles in the regulation of the phosphate (Pi) responsive signaling and metabolism (PHO) pathways, which keep the homeostasis of Pi in fungal cells. Except the PHO pathway, our recent results indicated that *VdNUC-2* participates a pathogenic regulation pathway which is indispensable for the penetration of host physical barriers.

**Methods and materials:** The wild-type strain V07DF2 was isolated from infected cotton in natural field. The original *VdNUC-2* mutant, named 6C4, was obtained by T-DNA random insertion. Other two *VdNUC-2* targeted deletion mutants, two ectopic complementation strains and three *VdNUC-1* targeted deletion mutants were introduced. VdNUC-1 is a crucial transcription factor located downstream of VdNUC-2, and it is positively regulated by VdNUC-2 in the PHO pathway. We observed cultural phenotypes of these strains under normal or low Pi culture condition, and accessed their virulence by employing the root irrigation method (intact root inoculation) or the root-dip method (wounded root inoculation). For further investigating the pathway(s) VdNUC-2 involved in, we performed yeast two-hybrid analysis and RNA-seq (total RNA isolated from inoculated plant roots).

**Results and conclusion:** ? All the strains grew normally on Czapek-Dox media with normal Pi content. However, compared with V07DF2 and the complementation strains, *VdNUC-2* and *VdNUC-1* mutants display growth defects under low Pi circumstances. ? The virulence of *VdNUC-2* mutants were significantly compromised on the cotton seedlings with intact roots, and that was unable to be restored by exogenous application of extra Pi. However, on the seedlings with wounded roots, the *VdNUC-2* mutants exhibit strong virulence and kill the hosts. Remarkably, the *VdNUC-1* mutants always displayed strong virulence as well as the wild-type strain regardless of inoculation method. These results proved that VdNUC-2 participates in at least two pathways, one is the PHO pathway, the other is pathogenic regulation pathway. ? Six proteins interacted with VdNUC-2 in yeast cells, and more than 800 genes were found with differential expression pattern in 6C4. Some of the differential expression genes play roles in cell wall hydrolysis, that might explain why the *VdNUC-2* mutants fail to penetrate the host physical barrier. For example, *endoglucanase EG1 gene* (VDAG_07825), encoding a Glycosyl hydrolase, has been proved to be a pathogenicity-related gene, and its log2-Ratio (6C4/V07DF2) are -1.85 (2 dpi) and -2.24 (5 dpi).

**548T Identification of an NB-LRR-encoding gene conferring sensitivity to victorin in *Phaseolus vulgaris*.** *Jenny Lorang*, Thomas Wolpert, Christina Hagerty Oregon State University, Corvallis, OR.

Breeding disease resistant plants is an essential, environmentally-favorable strategy for preventing crop loss due to disease. However, selection for resistance to one disease can inadvertently select for susceptibility to another. The species *Cochliobolus victoria* was described in 1946 as the casual agent of Victoria blight of oats but the pathogen was also associated with several weedy species, soybean, and barley. By the late 1940s Victoria blight of oat had reached epidemic proportions because susceptible oat genotypes had been widely planted as a source of resistance to the crown rust fungus *Puccinia coronata*. A dominant gene conditioning susceptibility to Victoria blight, *Vb*, was found to be genetically inseparable from the *P. coronata* resistance gene, *Pc2*. The *Vb* gene also was found to condition sensitivity to the *C. victoriae* toxin victorin. Recent studies of Arabidopsis, sorghum, barley and wheat suggest that necrotrophic pathogens are able to coopt resistance responses to cause disease. In this work we investigated the genetic basis of victorin sensitivity in *Phaseolus vulgaris*. We evaluated victorin sensitivity in Stampede x Red Hawk recombinant inbred lines (RILs) and identified a victorin sensitivity-associated QTL that contains Nucleotide Binding-Leucine Rich Repeat (NB-LRR) genes, a class of disease resistance (R) genes. One of these R genes conferred victorin-induced cell death when expressed in N. benthamiana and Arabidopsis. This gene is likely maintained in *P. vulgaris* for resistance functions, but could also be a source of necrotrophic disease susceptibility.

**549F The RNAi system in the Brassicaceae smut fungus *Thecaphora thlaspeos*.** *K. Boesch*¹,², L. Frantzeskakis¹, K. Courville¹, M. Feldbrügge¹,²,³, V. Göhre¹,²,³ 1) Heinrich-Heine-University, Düsseldorf, NRW, DE; 2) Bioeconomy Science Center (BioSC), Jülich, NRW, DE; 3) Cluster of Excellence on Plant Sciences (CEPLAS), Düsseldorf, NRW, DE.
Smut fungi infect important crop plants, e.g. maize or potato. To develop novel plant protection strategies, the infection process has to be characterized molecularly. Genetic manipulation of the crop host plants is still difficult because of their complex genomes. For this reason, we want to characterize the interaction of the smut fungus *Thecaphora thlaspeos* with the model plant *Arabidopsis thaliana*.

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

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

**550W Elucidating how Histoplasma capsulatum, a fungal intracellular pathogen, interferes with host cell biology. Rosa**

Rodriguez, Nancy Van Prooyen, Dervla Isaac, Anita Sil Dept. of Microbiology and Immunology, UCSF, San Francisco, CA.

*Histoplasma capsulatum* (*Hc*), a human intracellular fungal pathogen, subverts the normal biology of eukaryotic cells. *Hc* is a ubiquitous soil fungus that is aerosolized and inhaled into the mammalian lung when the soil is disrupted. Once in the lungs, *Hc* is phagocytosed by macrophages where it replicates within the phagosome by blocking acidification and maturation of this organelle through largely unknown mechanisms. The ability to colonize and lyse macrophages is thought to be a key step in successful infection. Our lab identified *Hc* insertion mutants that were defective in their ability to lyse bone-marrow-derived macrophages (BMDMs) during infection. One of these mutants harbors an insertion in the *SNX8* (sorting nexin 8) gene, so-named for its ortholog in mammalian cells. Sorting nexins (SNXs) are a family of proteins that are classified by the presence of phox-homology (PX) domains, which confer phosphoinositide (PtdInsP) lipid binding specificity. This lipid specificity recruits SNXs to PtdInsP rich early endosomes and SNXs are involved in a variety of cellular processes including membrane trafficking and remodeling. Our preliminary data indicate that the *Hc snx8* mutant is defective in manipulation of BMDMs. In culture, the *snx8* mutant has no growth defect; however, in BMDMs, it has a slight growth delay and fails to elicit host-cell lysis. Additionally, we used transmission electron microscopy (TEM) to compare the appearance of the phagosome during BMDM infection with either wild-type or *snx8* mutant *Hc*. We found that by 12 hours post infection (hpi), *snx8* mutants reside in a more spacious phagosome than wild-type *Hc*, indicating that the *snx8*-containing phagosome is fundamentally different from wild-type. We have also determined that the *snx8* mutant is avirulent in the mouse model of *Hc* infection, indicating that the inability of the *snx8* mutant to manipulate the host is detrimental to pathogenesis. We are using the *snx8* mutant as a tool to elucidate how wild-type *Hc* manipulates phagosome biology.

**551T A first glimpse into the genetic architecture of pathogenicity of Zymoseptoria tritici on bread wheat. T.C. MARCEL¹, A.**

DUCASSE¹, J. COMPAIN², N. LAPALU¹,², Z. ZHONG³, F. HARTMANN³, J. CONFAIS¹, H. GOYEAU¹, J. PALMA-GUERRERO³, G.H.J. KEMA⁴, J. AMSELEM², D. CROLL³ 1) BIOGER, INRA, AgroParisTech, Université Paris-Saclay, Thiverval-Grignon, France; 2) URGI, INRA, Université Paris-Saclay, Versailles, France; 3) Plant Pathology Group, ETH Zürich, Zürich, Switzerland; 4) Plant Research International B.V., Wageningen, The Netherlands.

*Zymoseptoria tritici* is a fungal pathogen of wheat responsible for the septoria leaf blotch disease. This disease is a major concern in bread and durum wheats growing areas worldwide. It can be controlled by fungicide treatments and the use of resistant wheat cultivars. To date, 21 resistance genes and 89 QTLs for resistance to septoria leaf blotch have been mapped in bread wheat. Despite this large availability of resistant sources, it remains a challenge to improve the resistance levels of wheat elite cultivars because of the mostly quantitative nature of wheat-septoria interactions and of the very high level of genetic diversity within pathogen populations. Our objective is to understand the genetic architecture of pathogenicity in *Z. tritici* by identifying its determinants and revealing the specificity of their interaction with resistance determinants in wheat. We have undertaken a genome wide association mapping approach (GWAS) to identify pathogenicity genes in *Z. tritici*. A collection of more than 2000 isolates has been established in France. The genetic diversity has been characterized in this collection, and 109 isolates selected for resequencing and pathogenicity assays. In so doing, we obtained the virulence spectra of the 109 French isolates, revealing the efficiency of known resistance *STB* genes in France, and allowing to detect known and unknown *STB* genes present in French elite cultivars. Furthermore, combining the precision of our pathology assays, the selection of wheat genotypes well characterized for their resistance determinants, and the precision of GWAS in our *Z. tritici* population, we have identified several candidate avirulence genes interacting with known resistance genes in bread wheat. Thus far, the small secreted protein *AvrStb6* is the first avirulence gene to be identified and functionally validated in this important wheat pathogen.

**552F A small secreted protein in Zymoseptoria tritici is responsible for avirulence on wheat cultivars carrying the Stb6**

Ziming Zhong¹, Thierry Marcel², Clémence Plissonneau¹, Bruce McDonald¹, Daniel Croll¹, *Javier Palma-Guerrero*¹ 1) Plant Pathology Group, ETH Zürich, Switzerland; 2) UMR BIOGER, INRA, , France.

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

**553W Construction and efficacy of Δcps1, a live attenuated vaccine for coccidioidomycosis.** *M. A. Mandel*¹,⁴, L. F. Shubitz¹, L. Lewis¹, H. T. Trinh¹, A. S. Buntzman², J. A. Frelinger³, J. N. Galgiani², M. J. Orbach¹,⁴ 1) The Valley Fever Center for Excellence, Tucson, AZ; 2) Department of Medicine, University of Arizona, Tucson, AZ; 3) Department of Immunobiology, University of Arizona, Tucson, AZ; 4) The School of Plant Sciences, University of Arizona, Tucson, AZ.

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

**554T A basidiomyceteous fungi-specific PsCaMKL1 encoding a CaMK-like protein kinase is required for the virulence and oxidative tolerance of Puccinia striiformis f. sp. tritici.** M Jiao, ZS Kang, J Guo State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi, P. R. China.

Calcium/calmodulin-dependent kinases (CaMKs) are Ser/Thr protein kinases (PKs) that respond to changes in cytosolic free $Ca^{2+}$ and play diverse roles in eukaryotes. In fungi, CAMKs are generally classified into four families CAMK1, CAMKL, RAD53 and CAMK-Unique. Among these, CAMKL is the largest family. In some fungal plant pathogens few CaMKLs have been shown to be responsible for pathogenesis, but little is known about their roles in rust fungi. In this study, we functionally characterized a novel PK gene, *PsCaMKL1*, from *Puccinia striiformis* f. sp. *tritici* (*Pst*). PsCaMKL1 belongs to a group of PKs that are evolutionarily specific to basidiomycetous fungi. It shows a highly conserved intra-species polymorphism and cytoplasmic localization in wheat protoplasts. *PsCaMKL1* is highly induced at early infection stages. The CaMK inhibitor KN93 blocked *Pst* ureidospore germination and suppressed transcription of *PsCaMKL1* during *Pst* germination. Overexpression of *PsCaMKL1* in fission yeast increased resistance to oxidative stress. Knock down of *PsCaMKL1* using host-induced gene silencing (HIGS) reduced the virulence of *Pst* accompanied by enhanced ROS accumulation and a hypersensitive response. These results suggest that *PsCaMKL1* is a novel and important factor that exerts its virulence function by regulating ROS production in wheat.

**555F Identification of novel classes of effector proteins of multicellular fungi against nematodes by challenge of vegetative mycelia with fungivorous nematodes in microfluidics devices.** *Annageldi Tayyrov*¹, Stefanie S. Schmieder¹, Aleksandr Goryachkin¹, Claire E. Stanley², Andrew J. DeMello², Markus Aebi¹, Markus Künzler¹ 1) Institute of Microbiology, Department of Biology, ETH Zürich, Vladimir-Prelog-Weg 4, CH-8093 Zürich, Switzerland; 2) Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg 1, CH-8093 Zürich, Switzerland.

Multicellular fungi defend themselves against antagonists that share a same growth environment with them. For this purpose, the non-motile fungi have developed several defense lines. In addition to physical defense lines, such as the buildup of melanized outer layers or chitinous cell walls, fungi mainly rely on chemical defense mediated by effector molecules (toxins). Characterized classes of fungal defense effectors include secondary metabolites, peptides and proteins. The best known examples of these classes are penicillin, alpha-amanitin and fungal pore-forming toxins/protease inhibitors/lectins, respectively. Most likely, this list of fungal defense effectors is not complete and, thus, novel approaches for the identification of such molecules are needed in order to explore the fungal defenseome. It has been shown that genes coding for fungal defense effectors can be identified on the basis of differential gene expression upon challenge with antagonists. Hence, in this study we conduct a genome-wide analysis of transcription (RNA sequencing) of the model mushroom *Coprinopsis cinerea* challenged by the fungivorous nematode *Aphelenchus avenae* at different time points in a tailor-made microfluidics device. Based on the expression profile of genes coding for already characterized anti-nematode effector proteins of *C. cinerea*, we set up a list of putative novel classes of anti-nematode effector proteins of this fungus. The list contained two types of cytoplasmic proteins with putative lipase domains that showed nematotoxicity when heterologously expressed in *Escherichia coli* and tested for toxicity towards the bacterivorous model nematode *Caenorhabditis elegans*. Site-specific mutagenesis of predicted catalytic residues reduced the toxicity significantly. Our results suggest that cytoplasmic lipases may constitute a novel class of fungal effector proteins against nematodes.

**556W Ancient horizontal transfer of a toxin biosynthesis cluster reveals novel mechanisms in the cercosporin biosynthesis pathway.** *Melvin Bolton*¹, Ronnie de Jonge², Malaika Ebert³, Bart Thomma³ 1) USDA - ARS, Fargo, ND; 2) Department of Plant Microbe Interactions, Utrecht University, Utrecht, The Netherlands; 3) Department of Phytopathology, Wageningen University, Wageningen, The Netherlands.

Cercospora leaf spot (CLS) caused by the fungus *Cercospora beticola* is considered to be the most destructive foliar disease of sugarbeet worldwide. The ability of the fungus to cause disease relies primarily on the production of a toxin called cercosporin during infection. Cercosporin is a photo-activated secondary metabolite toxic to a wide array of organisms including bacteria, mice, and plants but not to
cercosporin-producing *Cercospora* species. The cercosporin biosynthesis pathway has been well-characterized and thought to consist of eight cercosporin toxin biosynthesis (*CTB*) genes. We used phylogenomics to show that the *CTB* cluster is not limited to *Cercospora* species and has experienced an unprecedented number of duplications, losses, and horizontal transfers across a spectrum of plant pathogenic fungi during evolution. Since comparative genomic analysis revealed extensive gene collinearity adjacent to the established *CTB* cluster in all *CTB*-harboring species, we confirmed that *CTB* is larger than previously recognized and includes at least five additional genes, three of which are required for cercosporin biosynthesis. Additionally, the expanded cluster encodes the major facilitator superfamily transporter CFP, which contributes to cercosporin auto-resistance in *C. beticola*. Together, our results give new insight on the intricate evolution of the *CTB* cluster.

**557T Functional characterization of TvCyt2, a p450 monooxygenase relevant during plant-fungus interaction and mycoparasitism by Trichoderma virens.** Vianey Olmedo-Monfil, Daniela Porras-Troncoso, Claudia Ramírez-Valdespino, Alma Corrales-Escobosa, Kazimierz Wrobel

DCNyE Universidad de Guanajuato, Noria Alta S/N CP: 36050 Tel. (473) 7320006 ext. 8199 vg.olmedo@ugto.mx

In nature plants interact with harmful and beneficial organisms, in both cases the plant defense pathways can be activated through Microbe-Associated Molecular Patterns. In these interactions are also involved effector molecules, that can alterate structure and function of the host and are determinants in the type of association that is going to be established. The genus *Trichoderma* includes species of fungi widely known for its mycoparasitic activity. Furthermore, during its interaction with plants, activate defense mechanisms and promote plant growth and development, providing plant protection against infection by pathogens and abiotic stress. There is little information regarding *Trichoderma* effector molecules and their possible role in establishing the biological interactions. With the interest of identifying these molecules, we select 15 genes encoding possible effector type proteins, from a predicted secretoma of *Trichoderma*. Validation by qRT-PCR showed that three of these genes increased their expression level during interaction with the plant, while one of them, named *Tvcyt2*, showed a significant decrease on its expression. *Tvcyt2* encodes an enzyme classified as a monooxigenase P450. This kind of enzymes are involved in metabolic pathways related to detoxification processes and secondary metabolites synthesis. In order to determine the possible role of this gene in *Trichoderma*, we analyzed the metabolic profile obtained from overexpressing (OE TvCyt2) and null mutant (Δtvcyt2) strains, finding secondary metabolites present only in the OE TvCyt2 strain. The strains showing this modified metabolic profile had a better antagonistic activity against *Rhizoctonia solani* and activated with greater intensity defense pathways in *Arabidopsis*. Additionally, these strains promoted an increase in biomass and fitness in tomato plants. Our results indicate that TvCyt2 participates in the synthesis of secondary metabolite, which have a relevant role during plant-fungus interaction and mycoparasitism by *Trichoderma virens*.

Vianey Olmedo-Monfil, Daniela Porras-Troncoso, Claudia Ramírez-Valdespino, Alma Corrales-Escobosa and Kazimierz Wrobel.

DCNyE, Universidad de Guanajuato, Noria Alta S/N CP: 36050 Tel. (473) 7320006 ext. 8199 vg.olmedo@ugto.mx

In nature plants interact with harmful and beneficial organisms, in both cases the plant defense pathways can be activated through Microbe-Associated Molecular Patterns. In these interactions are also involved effector molecules, that can alterate structure and function of the host and are determinants in the type of association that is going to be established. The genus *Trichoderma* includes species of fungi widely known for its mycoparasitic activity. Furthermore, during its interaction with plants, activate defense mechanisms and promote plant growth and development, providing plant protection against infection by pathogens and abiotic stress. There is little information regarding *Trichoderma* effector molecules and their possible role in establishing the biological interactions. With the interest of identifying these molecules, we select 15 genes encoding possible effector type proteins, from a predicted secretoma of *Trichoderma*. Validation by qRT-PCR showed that three of these genes increased their expression level during interaction with the plant, while one of them, named *Tvcyt2*, showed a significant decrease on its expression. *Tvcyt2* encodes an enzyme classified as a monooxigenase P450. This kind of enzymes are involved in metabolic pathways related to detoxification processes and secondary metabolites synthesis. In order to determine the possible role of this gene in *Trichoderma*, we analyzed the metabolic profile obtained from overexpressing (OE TvCyt2) and null mutant (Δtvcyt2) strains, finding secondary metabolites present only in the OE TvCyt2 strain. The strains showing this modified metabolic profile had a better antagonistic activity against *Rhizoctonia solani* and activated with greater intensity defense pathways in *Arabidopsis*. Additionally, these strains promoted an increase in biomass and fitness in tomato plants. Our results indicate that TvCyt2 participates in the synthesis of secondary metabolite, which have a relevant role during plant-fungus interaction and mycoparasitism by *Trichoderma virens*.

**558F The role of BoiA, a Boi1-like protein, in fungal-plant symbiosis.** E. L. Bradley¹,², PY. Dupont¹,², R. Bradshaw¹,², A. Ram¹, B. Scott¹,², C. J. Eaton¹,² 1) Institute of Fundamental Sciences, Massey University, Palmerston North, NZ; 2) Bio-Protection Research Centre, Massey University NZ.

Reactive oxygen species (ROS) produced by the fungal NADPH oxidase (Nox) complex play an important role in regulating both mutualistic and antagonistic fungal-plant interactions. In the mutualistic symbiotic interaction between the fungal endophyte *Epichloë festucae* and its grass host *Lolium perenne*, loss of ROS signalling through disruption of *noxA* leads to a dramatic change in the host-interaction phenotype (Tanaka et al. 2006). Hyphae are defective in cell-cell fusion and undergo proliferative, instead of restrictive, growth in the leaves of the host. Plants infected with *ΔnoxA* are severely stunted and prematurely senesce. The fungal NoxA complex consists of membrane bound NoxA and NoxD components and cytosolic subunits NoxR, RacA, BemA and Cdc24, which are recruited to the membrane in response to some as yet unidentified signal. Activation of the mammalian Nox2 (gp91phox) complex involves changes in the cell membrane phosphoinositides, which leads to recruitment of the PX-domain cytosolic components, p47phox and p40phox, together with p67phox and Rac1. Although BemA contains a putative phosphoinositide-binding PX domain, this has been shown to be dispensable for membrane localisation in *Neurospora crassa* (Schürg et al. 2012). Instead, we hypothesize that recruitment of BemA occurs via interaction with the phosphoinositide-binding Boi1-like protein, which is known to interact with Bem1 in yeast. *E. festucae* contains two Boi1-like proteins, designated BoiA and BoiB. Phylogenetic analysis suggests *E. festucae* genes encoding these proteins arose from a common ancestor shared with *Saccharomyces cerevisiae* *BOI1* and *BOI2*. Transcriptome studies showed that *boiA* is highly expressed *in planta* compared to in culture, and up-regulated in mutants that disrupt the mutualistic interaction, suggesting an important role for *boiA* in symbiotic maintenance (Eaton et al. 2015). Phenotype analysis of *ΔboiA* deletion mutants revealed a loss of hyphal cell-cell fusion in culture compared to wild-type. Plants infected with *ΔboiA* were hypertillered and had a significant reduction in tiller length compared to the wild-type strain. Hyphae of *ΔboiA*, unlike wild-type, formed convoluted structures within the leaves, colonised vascular bundles and were frequently broken. These results show that BoiA is required for establishment of a mutualistic symbiotic interaction between *E. festucae* and *L. perenne*.

**559W Mollicutes-related endobacteria in the Mortierellomycotina.** A. Desirò¹, Z. Hao¹, GMN. Benucci¹, N. Vande Pol¹, Y. Chang², K. Barry³, I. Grigoriev³, JW. Spatafora², G. Bonito¹ 1) Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI; 2) Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR; 3) Department of Energy, Joint Genome Institute, Walnut Creek, CA.

Bacterial interactions with animals and plants have been examined for over a century; in contrast, the study of bacterial-fungal interactions has received less attention until recently. Bacteria and fungi interact at different levels and bacterial endosymbionts, which dwell inside fungal cells, provide the most intimate example. The most notable bacterial endosymbionts are associated with fungi belonging to the phylum Mucoromycota. The coenocytic nature of these fungi offers a niche for two types of endobacteria: *Burkholderia*- and Mollicutes/*Mycoplasma*-related endobacteria (MRE). MRE have been identified within the mycelium and spore of two out of the three Mucoromycota subphyla, the Glomeromycotina and Mucoromycotina; differently, MRE have never been reported in the Mortierellomycotina. We screened over 350 Mortierellomycotina strains for the presence of MRE and detected novel endobacteria phylotypes in nine of them.