Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSnumber of LysM domains, which are carbohydrate-binding modules. Ecp6, secreted by tomato leaf mould fungus Cladosporium fulvum, is the firstcharacterized LysM effector. We demonstrated that Ecp6 specifically binds chitin, the major constituent of fungal cell walls that acts as a microbialassociatedmolecular pattern (MAMP) and triggers immune responses upon recognition by the host. Ecp6 outcompetes plant receptors for chitin binding,and thus prevents the activation of immune responses. Many fungal genomes, including saprophytes, carry multiple LysM effector genes that share onlylow sequence conservation and encode a varying number of LysM domains. We speculate that fungal LysM effectors might bind different carbohydratesand exert various functions in fungal fitness. In the fungal wheat pathogen Mycosphaerella graminicola, two LysM effectors were identified. Mg3LysM, butnot Mg1LysM, suppresses chitin-induced immune responses in a similar fashion as Ecp6. Interestingly, unlike Ecp6, both Mg1LysM and Mg3LysM inhibitdegradation of fungal hyphae by plant chitinases, revealing an additional function for LysM effectors in pathogen virulence. We recently observed thatMg1LysM binds to the bacterial cell wall constituent peptidoglycan. Similarly, a LysM effector from the saprophytic fungus Neurospora crassa showedpeptidoglycan binding. We hypothesize that peptidoglycan binding by LysM effectors plays a role in the interaction of fungal species with bacterialcompetitors. The soil-borne fungal pathogen Verticillium dahliae contains seven LysM effectors genes of which one (Vd2LysM) is induced during tomatoinfection. Inoculation with two independent knock-out mutants revealed that Vd2LysM is required for full virulence of V. dahliae. However, Vd2LysM doesnot specifically bind chitin and does not function in a similar fashion as previous characterized LysM effectors. Thus, its function in virulence remainsunclear.523. WITHDRAWN524. Functional analysis and localization of SnTox1, a necrotrophic effector produced by the wheat pathogen Stagonospora nodorum. Zhaohui Liu 1 ,Weilin Shelver 2 , Justin Faris 3 , Timothy Friesen 1,3 . 1) Department of Plant Pathology, North Dakota State University, Fargo, ND; 2) USDA-ARS, BiosciencesResearch Laboratory, Fargo, ND; 3) USDA-ARS, Northern Crop Science Laboratory, Fargo, ND.SnTox1 is one of the necrotrophic effectors produced by the fungus Stagonospora nodorum, the causal agent of wheat Stagonospora nodorum blotch. Itinteracts, directly or indirectly, with the product of the wheat gene Snn1 to induce host cell death and promote disease. Previously, we showed thatSnTox1, a cysteine-rich protein, triggers programmed cell death-like responses in the host and plays an important role in fungal penetration. In the presentwork, we are investigating the biochemical and molecular function of SnTox1 as well as its mode of action. Based on a Prosite motif search of SnTox1,multiple predicted sites including a putative chitin binding domain were targeted for site-directed mutagenesis. SnTox1 activity was significantly reducedwhen mutations were produced at a casein kinase II phosphorylation site and a predicted helical region where lysine residues are abundant.Using a fungalstrain expressing an SnTox1-GFP fusion protein, we examined the location of the SnTox1 protein during fungal growth and infection. SnTox1 was observedin higher concentration on several fungal structures, including the surface of conidia and mycelium, hyphal septa, and hyphal tips. The accumulation ofSnTox1-GFP is particularly obvious at hyphal regions where new hyphae are arising. This observation suggests a protection mechanism of SnTox1 that issimilar to that of chitin binding proteins in other fungal pathogens. In planta, SnTox1 is highly expressed in the hyphopodia where the penetration isinitiated, providing further evidence that SnTox1 plays a role in penetration. The cellular localization of SnTox1 was also investigated using fluorescinelabeled SnTox1 in combination with cytological methods and preliminary data has indicated that SnTox1is likely not internalized into mesophyll cells butremains in the apoplast. Interestingly, SnTox1 is able to induce host cell death by directly spraying onto the leaf surface of sensitive lines. We are currentlyinvestigating if SnTox1 is transported through epidermal cell layer.525. Host-targeting protein 3 (SpHtp3) from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a pH and tyrosine O-sulfatedependentmanner. Lars Löbach 1* , Stephan Wawra 2 , Irene de Bruijn 2 , Aleksandra Toloczko 2 , Tim Rasmussen 3 , Christopher Secombes 1 , Pieter van West 2 . 1)Scottish Fish Immunology Research Centre, University of Aberdeen, School of Biological Sciences, Aberdeen, Scotland, UK; 2) Aberdeen OomyceteLaboratory, University of Aberdeen, School of Medical Sciences, Foresterhill, Aberdeen, Scotland, UK; 3) University of Aberdeen, School of MedicalSciences, Foresterhill, Aberdeen, Scotland, UK.The success of eukaryotic oomycete pathogens depends largely on effector proteins, molecules which manipulate or interfere with host defencemechanisms in the extracellular space or inside their host cells. One economical important oomycete parasite is the fish pathogen Saprolegnia parasitica,which is the causal agent of the disease Saprolegniosis. S. parasitica is responsible for devastating losses in the aquaculture industry worldwide. In order toeffectively fight any pathogen it is crucial to understand the key molecular mechanisms that lead to the disease. With the focus on putative effectorproteins we screened the genome of S. parasitica in the present study for potential effector candidates. Analysis identified a novel putative secreted S.parasitica effector protein, which we named host-targeting protein 3 (SpHtp3). Gene expression analyses showed that mRNA levels of SpHtp3 are highestin mycelium, sporulating mycelium and during the later stages of infection. Recombinant SpHtp3 was able to translocate specifically into fish cells in atyrosine O-sulfate and pH dependent manner. SpHtp3 was found in vesicular structures inside fish cells and was released from these upon infection of thecells with S. parasitica. Interestingly, SpHtp3 possesses an N-terminal RTLR tetra-peptide sequence at a similar location as found in RxLR-effectors fromplant pathogenic oomycetes. However, this RTLR-sequence was not required for the fish cell translocation property of SpHtp3. These findings suggest thatSpHtp3 from S. parasitica is a novel intracellular protein that might play an important role in Saprolegniosis.526. Ave1-like orthologs in Venturia: another expanded effector family emerges. Adam Taranto 1 , Daniel Jones 1 , Jason Shiller 1 , Shakira Johnson 1 , NathanHall 1 , Ira Cooke 1 , Gert Talbo 1 , Carl Mesarich 2 , Bart Thomma 2 , Jordi Boshoven 2 , Joanna Bowen 3 , Cecilia Deng 3 , Matthew Templeton 3 , Kim M. Plummer 1 . 1)Dept Botany, La Trobe Univ, Melbourne, Victoria, Australia; 2) Laboratory of Phytopathology, Wageningen University, The Netherlands; 3) Plant & FoodResearch, Auckland, New Zealand.Effectors are secreted by pathogens to modify plant physiology and establish disease. Plant immune receptors have evolved to recognise effectors andcounter attack with defence responses. Most fungal effectors are lineage-specific, i.e. they are unique to a species, or to physiological races within aspecies. The availability of many whole genome sequences has revealed that some effectors are found in a discontinuous distribution within the fungalkingdom; a few phytopathogenic fungi (Colletotrichum higginsianum, Cercospora beticola, Fusarium oxysporum) possess an ortholog of Ave1 fromVerticillium dahliae, an effector that activates Ve1-mediated resistance in tomato. A subset of these orthologs were shown to activate Ve1-mediatedresistance in tomato. Unusually, Ave1 also shares similarity to an ortholog in the phytopathogenic bacterium Xanthomonas axonopodis, as well as to awidespread family of plant natriuretic peptides and expansins, involved in plant homeostasis and plant cell wall modification (de Jonge & van Esse et al.2012). We have identified an expanded Ave1-like gene family in apple and pear scab fungi, Venturia inaequalis and V. pirina. These species also haveexpanded gene families with similarity to the Leptosphaeria maculans effector AvrLm6. V. pirina has 14 unique hits (best,1.43e -18 ) to VdAve1. V. inaequalishas 17 unique hits (best,1.07e -22 ) to VdAve1. The distribution of Ave1 orthologs is suggestive of one or more cross-kingdom gene transfer events. We arecharacterising Venturia Ave1-like genes to investigate the mode of gene multiplication; seek evidence of horizontal gene transfer; and determine the role250