Innate immunity of plants: basal defence and pathogen

counterdefence (IIP2012)

June 03-09, 2012 Heimari, Ristiina, Finland

Program and abstracts

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Innate immunity of plants: basal defence and pathogen counterdefence (IIP2012) J. Santala, E. M. Marttinen and J. P. T Valkonen (eds.)

Helsinki, Finland

ISBN 978-952-10-8066-1 (pdf)

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Programme Date Time Activity Teacher/responsible Sunday June 3

17:15 Departure from Helsinki airport Please look for the course assistants Mikko and Isabel in the arrivals hall. They will be there from 16:30 onwards.

Mikko Lehtonen and Isabel Weinheimer

20:00 Registration in hotel Heimari and taking luggage to the rooms

Personnel of Hotel Heimari

20:30

Dinner Information about practical issues

Jari Valkonen

Monday June 4

07:30 Breakfast 09:00 Presentation of participants Magnus Karlsson 09:45 L1 Introduction to innate immunity S. Dinesh-Kumar 10:45 Poster session 1 Jari Valkonen 12:45 Lunch Lectures Chair: Minna Pirhonen 14:00 L2 Autophagy. Basics of the mechanism S. Dinesh-Kumar 15:30 Coffee 16:00 L3 Role of autophagy in defence-

associated cell death Daniel Hofius

17:00 Studies in groups (A) S. Dinesh-Kumar & D. Hofius 19:00-20 Dinner 19:30-23 Sauna

Tuesday June 5

07:30 Breakfast 09:00 L4 The effector concept and

mycoparasitism Magnus Karlsson

10-12 Studies in groups (B) Dan Funck Jensen, Malin Elfstrand, Magnus Karlsson

12:00 Lunch 13:00 Excursion to Mikkeli (Ruralia Institute) Jari Valkonen 20:00 Dinner

Wednesday June 6

07:30 Breakfast Lectures Chair: Fred Asiegbu 09:00 L5 Bacterial effectors Minna Pirhonen 10:00 L6 Rice blast as a model system for host-

fungal pathogen interactions Yong-Hwan Lee

11:00 Break 11:10 L7 Innate immunity: bacterial microbe

associated molecular patterns (MAMPs), elicitors of plant innate immunity

Mari-Anne Newman

12:10 Lunch 13:30 Poster session 2 Jari Valkonen 15:30 Coffee Lectures Chair: David Collinge 16:00 L8 Involvement of ROS, a class III

peroxidase and the mitochondrial protein TSPO1 in the response of Physcomitrella patents to chitosan, a fungal elicitor

Mikko Lehtonen

16:25 L9 Lipopolysaccharide (LPS) and systemic responses in Arabidopsis

Jon Nielsen

Break Chair: Mari-Anne Newman 17:00 L10 Aphids suppress plant basal defense

responses Claire Drurey

17:25 L11 Dickeya solani: ecology, competition and functional genomcis

Linda Garlant

18:30 Dinner 19-23 Sauna

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Thursday June 7

07:30 Breakfast Lectures Dan Funck Jensen 09:00 L12 Functional dynamics of

phytopathogen effectors Sophien Kamoun

10:30 Break 10:45 L13 Functional analysis of glycoside

hydrolase family 18 and 20 genes in Neurospora crassa

Georgios Tzelepis

11:10 L14 Pathogenicity and host resistance in Heterobasidion-conifer tree pathosystem: challenges and perspectives in a genomic era

Fred Asiegbu

12:10 Lunch Lectures Chair: Yong-Hwan Lee 13:35 L15 Induced defence responses in

Norway spruce may involve an interaction between salicylic acid and jasmonic acid mediated signalling pathways

Miquel Nemesio Gorriz

14:00 L16 Pathogen defence in the rose family model plant Fragaria vesca

May Bente Brurberg

15:00 Coffee 15:30 Excursion Jari Valkonen 24:00 Return to Heimari

Friday June 8

07:30 Breakfast Lectures Chair: May Bente Brurberg 09:00 L17 Phosphosignalling and pathogenesis-

related (PR) proteins in plant immunity Erik Andreasson

10:00 L18 Time course analysis of leaf transcriptome after phosphite treatment for induced defence responses in Solanum tuberosum

Burra Dharanidhar

10:25 Studies in groups (C) Sophien Kamoun 12:30 Lunch Lectures Chair: S. Dinesh-Kumar 13:45 L19 dsRNA-tiggered immunity and viral

effectors Jari Valkonen

14:45 L20 Evolution of RNAi in Basidiomycota Yang Hu 15:15 Coffee 15:45 L21 Distribution of viruses inhabiting

Heterobasidion annosum in a pine-dominated forest plot in southern Finland

Rafiqul Hyder

16:10 L22 Engineering resistance to the plant pathogen Phytophthora infestans by manipulation of phytoalexin pathways

Artemis Giannakopolou

16:35 L23 Engineering pathogen resistance in crop plants

David Collinge

17:35 Course evaluation Jari Valkonen 19:00 Dinner, sauna, barbecue

Saturday June 9

07:30 Breakfast 09:00 Departure to Helsinki 11:45 Arrival at Helsinki airport

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Lecture Abstracts

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L1 Emerging perspectives on plant innate immunity S.P. Dinesh-Kumar Department of Plant Biology and The Genome Center University of California, Davis, CA 95616, USA As plants lack a global circulatory immune system, every plant cell must have the ability to recognize non-self molecules and mount a defense response. One branch of a plant’s immune system utilizes plasma membrane-localized receptors that recognize highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs). However, many pathogens are able to breach PAMP-triggered immunity and establish infection by injecting or secreting specific virulence proteins termed effectors into the host cell. To perceive these effectors, plants employ intracellular nucleotide binding-leucine-rich repeat domain containing intracellular immune receptors (NB-LRRs). These NB-LRR domains are also present in animal intracellular NOD-like receptors (NLRs) suggesting a conserved role in defense across kingdoms. There are two subclasses of plant NB-LRR receptors: CC-NB-LRRs have a coiled-coiled (CC) domain and TIR-NB-LRRs have a Toll-interleukin-1 receptor (TIR) homology domain. Notably, the TIR domain is also found in other important animal innate immunity proteins such as mammalian Toll-like receptors (TLRs). Although there are structural similarities between plant and animal NB-LRRs, the diversity and complexity of recognition mechanisms, associated factors, and activation observed with plant NB-LRRs appear to surpass their animal counterparts. I will discuss current perspectives on the molecular mechanisms by which immune receptors recognize pathogens and initiate immune signaling.

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L2 Role of autophagy in development, cell death, immunity, and beyond S.P. Dinesh-Kumar Department of Plant Biology and The Genome Center University of California, Davis, CA 95616, USA Macroautophagy, here after referred to as autophagy, is a dynamic process during which cytoplasmic materials are enclosed in double membrane-bound vesicles called autophagosomes that are then targeted to the vacuole or lysosome for degradation or recycling. The process of autophagy was first described more than fifty years ago; however, for many decades our understanding of autophagy was based largely on morphological observations from electron microscopy. Only in the past decade has the field expanded with discovery and molecular characterization of the yeast AuTophaGy-related (ATG) genes. Furthermore, the analysis of sequenced genomes of higher eukaryotes has identified ATG homologs in mammals, C. elegans, Drosophila, Dictyostelium, and plants, and many of these genes have been shown to be essential for autophagy function in these organisms. In addition to the identification of the ATG genes, significant progress has been made in the past decade in understanding some of the signaling events that regulate autophagosome formation and autophagy. It has long been known that autophagy’s recycling function is believed to be an important adaptive response to nutrient deprivation and other forms of environmental stress. However, recent studies have revealed that autophagy participates in other diverse biological processes including cellular differentiation and development, tissue homoeostasis, aging, leaf senescence, cell growth control, innate and adaptive immunity, cell death, and several diseases including cancer. I will discuss emerging perspectives on plant autophagy in relation to other organisms.

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L3 Role of autophagy in defence-associated cell death Daniel Hofius1, David Munch2, Qinsong Liu1, John Mundy2, Morten Petersen2 1Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, SE-75007 Uppsala, Sweden; 2Department of Biology, Copenhagen University, Ole Maaloes Vej 5, Copenhagen 2200, Denmark Autophagy is a conserved intracellular pathway for bulk degradation of cellular content or selective clearance of unwanted organelles, proteins and lipid, thus regulating cellular homeostasis. In addition, autophagic mechanisms facilitate survival during stresses such as starvation and microbial infections, but also promote programmed cell death (PCD) in certain developmental and pathological situations across the eukaryotic kingdoms. Consistent with well-established roles of autophagy in mammalian innate immunity, emerging evidence suggests that autophagic processes in plants are engaged in immune responses against different pathogens. In this context, we found that autophagy serves survival functions in basal defences against virulent microbes but also contributes to hypersensitive response-associated cell death triggered upon recognition of pathogen effectors by resistance proteins. However, the analysis of more specific functions of autophagy in pro-survival and pro-death decisions of the immune system seems to be complicated by the concurrent engagement of autophagy in homeostatic processes to protect from defense associated stress at the endoplasmatic reticulum (e.g. unfolded protein response). These observations together with recent concerns about the availability of real autophagy-null mutants in plants may also explain some of the discrepancies in the proposed roles of autophagy in basal immunity and defensive PCD. Here, we try to reconcile the opposing findings and present our current approaches to unravel novel regulators of immunity-associated autophagy and cell death.

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L4 The effector concept and mycoparasitism Magnus Karlsson1, Chatchai Kosawang2, David B. Collinge2, Jan Stenlid1, Dan Funck Jensen1. 1Forest Mycology and Plant Pathology, SLU, Swedish University of Agricultural Sciences, Box 7026, 75007 Uppsala, Sweden; 2Plant Biology and Biotechnology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. The concept of effector biology of plant-associated microbes suggests that microbes secrete proteins and other molecules, collectively known as effectors, which modulate plant defence and enable successful colonization of plant tissues. Here we expand the effector concept to fungal/fungal interactions and use it as a framework to study mechanisms of mycoparasitism in Trichoderma and Clonostachys fungal biocontrol agents. In Trichoderma, whole-genome sequencing reveal between 20 and 36 different genes encoding chitinases. Maximum likelihood-based evolutionary analyses reveal that chitinases chi18-13 and chi18-15 evolve in a manner consistent with rapid co-evolutionary interactions and identify putative target regions involved in determining substrate-specificity and enzyme architecture. Our results suggest that fungal/fungal interactions can drive adaptive changes in enzymatic properties as a response to specific ecological contexts of different Trichoderma species. Another mycoparasitic fungus, C. rosea, was previously shown to possess the ability to detoxify the Fusarium mycotoxin zearalenone (ZEN), through the enzyme zearalenone lactonohydrolase. We show that the zearalenone lactonohydrolase gene, zhd101, is induced 887-fold by 10 ppm ZEN, but repressed 5-fold by 5 g/l glucose or sucrose. A zhd101 disruption mutant is unable to degrade ZEN, and fail to protect wheat seedlings against F. graminearum Foot Rot disease in growth chamber tests. These results show that production of, and resistance to, secondary metabolites is an important factor in fungal/fungal interactions.

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L5 Bacterial effectors Minna Pirhonen Department of Agricultural Sciences, University of Helsinki Plant pathogenic organisms produce molecules that are recognised by the host. The recognition may lead to activation of the host defence mechanisms that are able to prevent the infection. A successful pathogen needs to suppress the host’s immune responses during the infection. Most Gram-negative bacteria posses a specific secretion mechanism, Type III secretion system (T3SS), that targets the host defence mechanisms during infection. Pseudomonas syringae is a model organism for the characterisation of T3SS secretes proteins, so called effectors. It secretes the proteins through the host cell wall into the host cell, where they target various functions, such as host transcription, chromatin structure and recognition processes. The combined effector functions lead to suppression of the host defence mechanism and progression of the disease. In this presentation, examples of T3SS effector functions in various plant pathogenic bacteria and new, emerging effector molecules are described.

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L6 Rice blast as a model system for host-fungal pathogen interactions Yong-Hwan Lee Departments of Forest and Agricultural Sciences, University of Helsinki, Helsinki, Finland; Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Center for Fungal Pathogenesis, Seoul National University, Seoul 151-921, Korea Rice blast, caused by Magnaporthe oryzae, is a compelling model system for studying host–parasite interactions due to its socioeconomic impact and the availability of both the rice and fungal genomic sequences. In an attempt to understand the molecular mechanisms of rice blast, we have been taking both forward and reverse genetics approaches. Our researches using reverse genetics approach focus on identifying and characterizing the genes involved in signal transduction pathways leading to appressorium formation, genes encoding transcription factors, and genes that are required for post-penetration stages. For forward genetics studies, we carried out a large-scale insertional mutagenesis of the M. oryzae strain KJ201 via Agrobacterium tumefaciens-mediated transformation, generating over 25,000 mutants. We also developed high throughput phenotype screening system that enables rapid and robust assay of mutant phenotypes. Those mutants are stored and maintained in the Center for Fungal Genetic Resources. In addition to our endeavor to functional and comparative genomics, we built a cyber-infrastructure for storage of heterogeneous data and analysis of such data in multiple contexts. The whole genome sequence information of M. oryzae as well as most of the results from experimental biology is housed in our customized database. Our comprehensive and integrative approaches coupled with a web-based Laboratory Information Management System would provide a novel platform for systems biology initiatives for fungal pathogenesis.

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L7 Innate Immunity: Bacterial Microbe Associated Molecular Patterns (MAMPs), Elicitors of Plant Innate Immunity Mari-Anne Newman Faculty of Science, Department of Plant Biology & Biotechnology, University of Copenhagen, Denmark. Email: mari@life.ku.dk In an environment that is rich in potentially pathogenic microorganisms, the survival of higher eukaryotic organisms depends on efficient pathogen sensing and rapidly mounted defence responses. Such protective mechanisms are found in all multicellular organisms and are collectively referred to as innate immunity. Plants interact with a variety of microorganisms, and like insects and mammals, they respond to a range of microbial molecules from both host and non-host pathogens. The elicitors are essential structures for pathogen survival and are for that reason conserved among pathogens. These conserved microbe-specific molecules, also referred to as Microbe or Pathogen Associated Molecular Patterns (MAMPs or PAMPs), are recognised by the plant innate immune systems Pattern Recognition Receptors (PRRs). We have chemically and biologically examined the MAMP activity of lipopolysaccharide (LPS) and peptidoglycan (PGN) from two Gram-negative plant pathogens. I will show that Arabidopsis PEN1, a SNARE protein believed to be required for docking and fusion of intracellular transport vesicles, is involved in signal transduction leading to the induction of the innate immune responses by particular bacterial MAMPs. Finally I will present data showing that two lysine motif (LysM) containing plasma membrane proteins, LYM1 and LYM3 from Arabidopsis, interact with PGN; and that the transmembrane LysM receptor kinase CERK1 is involved in transmembrane signaling.

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L8 Involvement of ROS, a class III peroxidase and the mitochondrial protein TSPO1 in the response of Physcomitrella patens to chitosan, a fungal elicitor Mikko T. Lehtonen,1 Motomu Akita,1,2 Wolfgang Frank,3 Ralf Reski,3,4 and Jari P.T. Valkonen1

1Department of Agricultural Sciences, University of Helsinki, Finland; 2Department of Biotechnological Sciences, Kinki University, Japan; 3Plant Biotechnology, Faculty of Biology, University of Freiburg, Germany; 4BIOSS Centre for Biological Signalling Studies, University of Freiburg, Germany Physcomitrella patens (wild type, WT) and selected gene knockout mutants were studied for their biotic stress responses. Chitosan treatment caused an instant oxidative burst of extracellular superoxide in P. patens WT. In contrast, the previously characterized mutant

Prx34 lacking the gene for the chitosan responsive secreted class III peroxidase (Prx34) resulting in an increased susceptibility to fungal infection (Lehtonen et al. 2009, New Phytol. 183: 432–443) was incapacitated in superoxide production. The genes encoding NADPH oxidase (NOX), alternative oxidase (AOX), plastidial lipoxygenase (LOX7), and a translocator protein TSPO1 involved in mitochondrial tetrapyrrole transport and abiotic stress were induced in chitosan treated P. patens WT. However, in the PpTSPO1 mutant (Frank et al. 2007, Plant J. 51: 1004–1018) expression levels of PpAOX, PpLOX7 and PpNOX were constitutively elevated and superoxide production upon chitosan treatment was doubled compared to P. patens WT. Unexpectedly, the PpTSPO1 mutants were more sensitive to fungal infection than WT suggesting uncontrolled damage by reactive oxygen species. Our results show that moss responds to a common pathogen elicitor with an oxidative burst, which involves a class III peroxidase, similar to vascular plants. Our results also indicate a role of mitochondrial PpTSPO1 in maintenance of redox homeostasis during biotic stress besides its known involvement in abiotic stress adaptation.

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L9 Lipopolysaccahride (LPS) and systemic responses in Arabidopsis. Jon T. Nielsen, Gitte Erbs, Mari-Anne Newman University of Copenhagen, Faculty of Science, Frederiksberg, Denmark jonthoe@life.ku.dk Plants benefits from a highly evolved innate immunity, which is the first line of defence to any invading microbe. A number of microbe-associated molecular patterns (MAMPs) are known, however only a few plant receptors (PRRs) for the MAMPs are described. One known MAMP is LPS from the outer membrane of Gram-negative bacteria. Though the PRR(s) recognising LPS are not described; LPS has been reported to have a number of effects on the induction of immune responses in plants (reviewed by Erbs & Newman 2012). After treatment of Arabidopsis leaves with labelled Salmonella Minnesota LPS, the LPS was found in treated as well as in systemic leaves after 24 hours (Zeidler et al., 2010). This contrast the results reported by Gross et al. (2005), where no intracellular accumulation of LPS could be detected. We want to investigate if LPS is systemically spread throughout the plant tissue and have labeled Xanthomonas campestris pv. campestris (Xcc) LPS with Alexa-488 (a fluorescent dye). We show that Xcc LPS spreads systemically throughout the plant tissue, and that the LPS fairly quickly (within an hour) is found only in the vascular system of the leaves. The observation of systemic spread of LPS and localization in the vascular system raises several questions: 1) How is LPS transported across membranes? 2) Is a measurable innate immune response induced by LPS in the systemic leaves? or 3) is the response in the systemic leaves a potentiation of defence as have been described in several system (Conrath, U. et al., 2006). We will attempt to answer some of these questions in this Master project. After the movement and the localisation of LPS have been fully established, a microdissection fluorescence microscope will be used to cut out and isolate single cells. Gene induction in the selected cells will be investigated further by PCR testing prepared cDNA with primers designed to already known genes in plant defence. Additionally cDNA will be prepared for microarray analysis to identify “new” genes involved in innate immunity. Conrath, U. et al., 2006. Priming: getting ready for battle. MPMI: 19, 1062 Erbs, G. and Newman M-A. 2012. Mol. Plant Pathol. 13: 95-104 Gross, A. et al., 2005. New Phytol. 165, 215–226 Zeidler, D.et al., 2010. Mol. Plant Pathol. 11, 747–755.

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L10 Aphids suppress plant basal defense responses Claire Drurey1, David Prince1, Saskia A. Hogenhout1

1 Department of Cell & Developmental Biology, The John Innes Centre, Norwich Research Park, Norwich, UK. Aphids are sap-feeding insects of plants that cause significant losses in crop yield. They do this both as a direct drain on plant resources by feeding from the phloem as well as acting as virus vectors. Due to these negative effects on plants it would be expected that plants have developed defenses against them. Basal plant defense responses such as PTI (PAMP triggered immunity) effectively fend off the majority of plant pathogens. Pathogens that produce virulence proteins (effectors) that suppress basal plant defenses can successfully colonise plants. So far, most research has focussed on microbial pathogens and it is not yet clear whether plants have basal defense responses to insect pests such as aphids. We found that aphids can evoke typical PTI responses such as calcium and ROS bursts and callose deposition. Furthermore, we have found at least one aphid effector that suppresses the ROS burst. Thus basal immunity is also a factor in plant defense to insect pests.

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L11 Dickeya solani: ecology, competition and functional genomics Linda Garlant, Minna Pirhonen. University of Helsinki Several species of plant pathogenic Gram-negative enterobacteria in the genus Dickeya are the causal agents of diseases in numerous plant species in tropical and warm climates and recently, Dickeya strains have been found even in temperate and cool regions in northern Europe. Dickeya solani is a new potato pathogen that has moved from ornamental plants to potato in Holland and now it spreads fast and replaces the closely related but less virulent potato pathogens in Europe. It seems to spread with Dutch seed potato, and in Netherlands it has caused five-fold increase in economical losses in potato production. Even in Finland the losses caused by these bacteria have increased consistently in the last few years. Symptoms caused by D. solani resemble typical blackleg symptoms (previously associated with other bacteria) and include wilting and rotting of plants and tubers, but the disease appears to be more aggressive and causes damage under a wider range of conditions and at lower bacterial loadings. Furthermore, the bacterium is able to spread from soil into plant roots and vascular tissue very efficiently, which causes a high level of spreading during growing season. Since the new Dickeya pathogen appears to be highly aggressive especially during warm summers, there are implications for increased importance of this pathogen in response to global warming. The entire genome sequence of one D. solani strain has been sequenced and its content analysis suggests major difference between this bacterium and the ones it is replacing, for example it has more genes coding for non-ribosomal peptide synthetases needed for the production of small molecules affecting competition or symbiosis with other soil organisms. Furthermore in vitro essays showed that D. solani is able to secrete some molecules that inhibit the growth of closely related potato pathogenic bacteria. Molecular characterization of bacterial competition in vitro and inside the host plant allows us to study the bacterial communication mechanisms at genetic level and to better understand why and how D. solani is competing with the other bacterial species and taking over in potato.

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L12 Functional dynamics of phytopathogen effectors Sophien Kamoun The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, United Kingdom The field of plant-microbe interactions has now coalesced around a general model. The major classes of molecular players both from plants (surface and intracellular immune receptors) and microbes (PAMPs and effectors) have now been revealed. This model applies to a diversity of plant pathogens, including bacteria, fungi, oomycetes, nematodes and insects, and is also relevant to symbiotic interactions. This presentation focuses on effectors, secreted microbial molecules that alter plant processes and facilitate colonization. Effectors are central to our newly integrated view of plant-microbe interactions. Effectors have evolved to facilitate parasitism for example by suppressing host immunity in a variety of ways. But they can also “trip on the wire” and activate plant immune receptors, a response known as effector-triggered immunity. These are complex interactions and the co-evolutionary dynamics between plants and microbes have left striking marks in both pathogen and plant genomes. I will discuss how effectors have evolved and adapted to their host cells to perturb plant processes. I will report on recent advances and highlight the most important open questions in effector biology.

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L13 Functional analysis of glycoside hydrolase family 18 and 20 genes in Neurospora crassa Georgios D. Tzelepis1, Petter Melin2, Dan Funck Jensen1, Jan Stenlid1 and Magnus Karlsson1 1Uppsala BioCenter, Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, 75007 Uppsala, Sweden and 2Uppsala BioCenter, Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, 75007 Uppsala, Sweden. Glycoside hydrolase (GH) family 18 and 20 contain chitinolytic enzymes responsible for chitin degradation. GH18 are phylogenetically divided into 3 groups (A, B and C), each further divided into subgroups. Subgroup B5 genes encodes enzymes with ENGase deglycosylation activity. In this study, we investigated the functional role of 10 N. crassa genes coding for chitinases, 2 genes encoding ENGases and 1 gene encoding a NAGase, using gene disruption and qPCR techniques. The ENGase disruption mutant ”gh18-10 showed slower growth rate on carbon rich media and on chitin plates, while it grew faster than WT during abiotic stress conditions. gh18-10 was constitutively expressed during growth on carbon rich media, during carbon starvation conditions, on chitin plates and during fungalfungal interactions. The function of gh18-10 may be connection with the endoplasmatic reticulum associated protein degradation process (ERAD). The two C2 subgroup chitinase genes gh18-6 and gh18-8 were both induced during fungalfungal interactions. However, gh18-6 was only induced during interspecific interactions, while gh18-8 displayed the highest expression levels during selfself interactions. gh18-8 also displayed a unique domain structure including 2 transmembrane domains, indicative of cell wall localization. These data suggest functional differentiation of N. crassa C2 chitinases; gh18-6 may function in aggressive interspecific interactions while gh18-8 may be a cell wall modifying enzyme

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L14 Pathogenicity and host resistance in Heterobasidion-conifer tree pathosystem: challenges and perspectives in a genomic era Fred O. Asiegbu Department of Forest Sciences, University of Helsinki, Helsinki, Finland Host–pathogen interactions have been studied mostly in agricultural crops, with very little work done in forest trees, particularly gymnosperms. Molecular and genomics studies in forest tree pathosystem lacks far behind parallel work in agricultural crop pathology. Due to the paucity of our knowledge about interactions in forest tree pathosystems, much of the information has been drawn with reference to agricultural systems. Although gymnosperms and angiosperms diverged during evolution several million years ago, a lot can be learnt from agricultural crop pathosystems. In tree pathosystems, much of the basic molecular research has mostly been conducted using Heterobasidion annosum as a model. However, although the biology and genetics of this most economically important conifer pathogen Heterobasidion annosum sensu lato has been studied, knowledge about mechanisms of defence responses, resistance and pathogenicity remains largely unexplored. A major set back for the conifer pathosystem is the lack of a suitable model system and DNA transformation has only been achieved in very few fungal pathogens of forest trees. Additionally, the long life cycle, size of the mature trees and long time-scale of many of their diseases make working with these plants inherently difficult. Equally, from the pathogen perspective, there are no avirulent strains of the pathogen and absence of any host genotype in the Pinaceae with total resistance against H. annosum further complicates detailed molecular analysis of the interaction. However, despite the drawbacks, a number of genome sequencing projects of several tree pathogens are currently ongoing or have been completed including that of Heterobasidion sp. It is expected that a novel combination of molecular and genomics approach will advance our knowledge of this pathosystem that will have direct relevance to conifer trees. This will obviously form the basis for resistance research as well as for increasing tree resistance through selection or genetic engineering.

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L15 Induced defence responses in Norway spruce may involve an interaction between salicylic acid and jasmonic acid mediated signalling pathways. Miguel Nemesio Gorriz1, Jenny Arnerup1, Karl Lundèn1, Fred Asiegbu2, Jan Stenlid1, Malin Elfstrand1 1Department of Forest Mycology and Plant Pathology, SLU; 2 Department of Forest Sciences, University of Helsinki. Norway spruce [Picea abies (L.)Karst.], a key tree species for forest industry in Europe, is a host for Heterobasidion annosum (Fr.) Bref. Sensu lato, which causes root and stem rot and is one of the major plagues for this tree species (Dalman et al. 2010). Previous work carried out by Arnerup et al. (2011) showed that inoculation with H. parviporum (Fr.) Niemelä & Korhonen induces genes involved in the salicylic acid (SA)- and jasmonic acid/ethylene (JA/ET)-mediated signaling pathways simultaneously. In the present study we attempt to dissect the background to the simultaneous induction of these supposedly antagonistic pathways. Bark samples were inoculated with H. parviporum , the saprotrophic fungus Phlebiopsis gigantea and wounded to study the tempo-spatial distribution of the responses. In an independent experiment seedlings were treated with a range of inhibitors and activators of the different pathways in the presence or absence of H. parviporum. Transcript accumulation patterns of genes representing the different pathways (SA; PR1, LURP1, and NPR1 and JA/ET; LOX, JAR1, JAZ1, MYC2, and ACS) were analyzed with quantitative PCR. A significantly higher induction of genes represented JA/ET and SA signalling was observed after fungal inoculation compared to wounding. In seedlings, MeSA and MeJA treatments both induce PR1 and LURP. We will show how the different inhibitors induce these defense pathways and how these pathways are related. References Arnerup, J., Lind, M., Olson, Å., Stenlid, J. & Elfstrand, M., 2011 The pathogenic white-rot fungus Heterobasidion parviporum triggers non-specific responses in the bark of Norway spruce. Tree Physiology 31/11, 1262-1272. Dalman, K., Olson, A., Stenlid, J. 2010. Evolutionary history of the conifer root rot fungus Heterobasidion annosum sensu lato. Molecular ecology 19/22, 4979-4993.

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L16 Pathogen defense in the rose family model plant Fragariae vesca May Bente Brurberg1, Jahn Davik2, Erik Lysø1, Muath Alsheikh3, Abdelhameed Elameen1, Inger Martinussen4, Jens Rohloff5, Per Winge5, Sonja Klemsdal1 1Bioforsk - Norwegian Institute for Agricultural & Environmental Research, Plant Health & Plant Protection, Ås, Norway; 2Norwegian Institute for Agricultural & Environmental Research, Kvithamar, Norway; 3Graminor Breeding Ltd., Ridabu, Norway; 4Norwegian Institute for Agricultural & Environmental Research, Tromsø, Norway; 5Norwegian University of Science and Technology, Trondheim, Norway. Strawberry is the most important fruit crop in Norway, but each year strawberry producers experience serious economic losses due to plant diseases caused by fungal or oomycete pathogens. We address two such diseases in our research, strawberry crown rot, caused by the oomycete Phytophthora cactorum, which is characterized by wilting and eventually collapse of the plant and strawberry powdery mildew caused by the fungus Podosphaera aphanis. An efficient and environmental friendly control measure is the use of resistant cultivars, however most commercial cultivars are susceptible to these diseases. Plant resistance to disease is mediated by a multilayered innate immune system governed by a magnitude of different genes. The aim of our work is to generate basic knowledge about plant resistance as well as to develop genetic markers that can be used as tools for development of resistant cultivars. The genetic complexity of the octoploid cultivated strawberry (Fragaria x ananassa Dutch.), has led to development of the diploid wild strawberry (F. vesca) as a model system for Fragaria. We have identified and characterized resistance genes from diploid strawberry using nucleotide-binding site (NBS)-profiling for resistance genes and microarray analysis, and we are currently working on resistance gene and marker discovery using next-generation sequencing.

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L17 Phosphosignalling and pathogenesis-related (PR) proteins in plant immunity Marit Lenman, Svante Resjö, Ashfaq Ali, Scott C Peck, Erik Andreasson Swedish University of Agricultural Sciences, Department of Plant Protection Biology Interactions between plants and microbes involve dynamic changes on RNA, protein and post translational levels. A classic outcome of these interactions is accumulation of Pathogenesis-related (PR) proteins. These include PR-1, a protein of unknown function; PR-2, -1,3-glucanases; PR-3, Chitinases I, II, IV, V, VI, VII; PR-4 Chitinases I, II; PR-5 Thaumatin-like proteins; PR-6 Proteinase-inhibitors, PR-7 Endoproteinases, PR-8 Chitinase III; PR-9 Peroxidases; PR-10 Ribonuclease-like; PR-11 Chitinases I; PR-12 Defensins; PR-13 Thionins; PR-14 Lipid-transfer proteins; PR-15 and PR16 Oxalate oxidases. One example of the use of these proteins as markers for defense responses is from a study of a paranoid potato clone that expresses PR1 constitutively without any apparent cost of the defense. However, normally before PR proteins are induced, a network of maybe redundant signaling takes place. Protein phosphorylation is involved in basically all eukaryotic cell biological processes. Examples of phosphoproteins that are involved in innate immunity are the flagellin receptor, WRKY transcription factors and Mitogen-Activated Protein Kinases (MAPKs). The specificity of MAPK-signaling is not well understood in plants. Although some substrates of MAPKs have been identified, no information is available about whether other amino acids in the primary sequence other than proline-directed phosphorylation contribute to kinase specificity towards substrates. We have searched for consensus phosphorylation sequences for MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted, and MPK3 and MPK6 phosphorylated all tested proteins more efficiently than MPK4. These results indicate that the amino acid residues in the primary sequence surrounding the phosphorylation site of MAPK substrates can contribute to MAPK specificity. Using qualitative and quantitative proteomics, we have identified more than 2000 different phosphorylation sites in Phytophthora infestans. Among the identified phosphoproteins are proteins involved in infection such as members of the CRN-family of effectors.

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L18 Time course analysis of leaf transcriptome after phosphite treatment for induced defence responses in Solanum tubersoum (cv. Des) B Dharanidhar (1), Therese Bengtsson (1), Laith Moshaib (1) , Pete Hedley (2) , Erland Liljeroth (1), Erik Andreasson (1), Erik Alexandersson (1)

1 Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden 2 James Hutton Institute, Invergowrie, Dundee Research on alternative sources of resistance in potato against the oomycete Phytophthora infestans is being actively pursued. One of the alternatives in induced resistance – which is an increased defence in plants after challenge with elicitor molecules. Field trials by us and others using Phosphite (Phi) has shown resistance against the oomycete. Even though many have attributed this mechanism of Phi to its inherent fungicidal/fungistatic property, its role in induction and activation of immune response and thereby leading to resistance cannot be ruled out. Our study is designed to identify the role of Phi in induced defence response. Proalexin (a commercially available potassium phosphite compound) was applied to the leaves of P. Infestans susceptible Solanum tubersoum (cv. Des) and currently its effects on transcriptome (agilent arrays based on 3.4V Solanum phureja – genome) is being studied in relation to induced defence and P. infestans resistance. Leaves were sampled at five different time points (3, 6, 11, 24 and 48 hrs) post Phi treatment. In parallel we have also conducted detached leaf assays at all-time points to correlate the changes in the leaf transcriptome to the observed resistance. Our initial results with the detached leaf assays demonstrate the ability of proalexin to reduce symptoms associated with oomycete infection, we also show persistence of resistance even on the second day after proalexin application. In relation to induced defence response, we are in preparation to study the effect of Phi on Potato Blackleg disease caused by Pectobacterium atrosepticum.

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L19 dsRNA-tiggered immunity and viral effectors Jari Valkonen Department of Agricultural Sciences, PO Box 27, FI-00014 University of Helsinki, Finland Plant cells recognise double-stranded RNA (dsRNA) specifically and target it to degradation. Two cellular pathways are used for the purpose. Viruses have evolved various means of evade or suppress antiviral defense in their host plants. For this purpose they produce effector proteins. The study on the mechanisms and targets of viral effector proteins reveals novel aspects of the antiviral defence system in plants. One of the most studied viral effectors is the RNA silencing suppressor protein HCpro of potyviruses, the largest group of plant-infecting RNA viruses. HCpro interacts with the translation initiation factors eIF4E and eIF(iso)4E for as yet unknown reasons, but the interaction is needed for infection. Host plants containing mutated forms of the eIF4E or eIF(iso)4E genes can escape the infection. Further understanding of the protein complexes formed by viral and host proteins is expected to raise new possibilities to develop virus-resistant plant cultivars.

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L20 Evolution of RNAi in Basidiomycota and its application as a biotechnology tool to study plant pathogen interactions. Yang Hu, Vélëz, Heriberto, Jan Stenlid, Malin Elfstrand, Åke Olson Department of Forest Mycology and Plant Pathology, SLU, Box 7026, SE-750 07 Uppsala, Sweden RNA interference (RNAi) is a conserved eukaryotic gene silencing phenomenon mediated by small (20–30 nucleotides) noncoding RNA (sRNA) molecules, which primarily are categorized to short interfering RNAs (siRNAs) and microRNAs (miRNAs). RNAi has three major functions: genomic defence, heterochromatin formation, and gene regulation. Since the phenomenon of RNAi was first discovered in the nematode Caenorhabditis elegans, accumulating evidence has indicated that two core components, Dicer and Argonaute, are important in RNAi machinery. Comprehensive phylogenetic analyses of Basidiomycetes genes encoding Argonaute and Dicer can be performed to gain insights into the diversification of RNA silencing pathways during the evolution of Basidiomycota. A deeper understanding of the evolution and mechanism of RNAi in the Basidiomycetes will be useful for application of this biotechnological tool to study plant fungal interactions and virulence factors of plant pathogens. As a tool in reverse genetics, RNAi was has been applied in more than 40 species of fungi for modification of gene expression. Heterobasidion annosum sensu lato (s.l.), which causes rot and butt rot to conifer, is the most destructive pathogen in boreal and temperate conifer forests of the northern hemisphere. The genome of one member of the species complex H. irregulare has been sequenced at Joint Genome Institute, JGI, USA. This allows for a direct use of sequence information for gene discovery and functional analysis. Guantitative trait locus (QTLs) for virulence were identified four, and positioned them on a genetic linkage map. By placing the QTLs on the physical sequence of the genome, about 418 candidate virulence factor gene models have been identified. Searching the H. irregulare and other Basidiomycte genome sequence available identified genes encoding both Argonaute and Dicer. Results on the phylogeny of Basidiomycetes Argonaute and Dicer will be presented. This information combined with available transformation method provides the ability of applying RNAi by expression of hairpin dsRNA in H. annosum s.l. to study candidate virulence factor.

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L21 Distribution of viruses inhabiting Heterobasidion annosum in a pine-dominated forest plot in southern Finland Rafiqul Hyder, Eeva Vainio, Tuula Piri, Heikki Nuorteva, Jarkko Hantula Finnish Forest Research Institute (METLA), Vantaa, Finland Abstract: The economic losses to the forest sector in Finland as well as in the whole Europe is due to Heterobasidion spp. fungi infections. These root-rot diseases spread vegetatively from tree to tree via root contacts and by basidiospores which infect freshly cut stump surfaces. Traditional control methods include avoiding summer time logging, tree species rotation, stump removal and biological or chemical stump treatments. However, none of these methods can fully eradicate the fungus from a contaminated site and therefore alternative methods are urgently needed. Mycoviruses sometimes confer beneficial or harmful effects to their host fungi. In this study, we screened the presence of putatively novel viruses inhabiting Heterobasidion annosum in a 1.5 ha pine forest plot in southern Finland. Nine dsRNA virus infections were detected among fifty H. annosum isolates using CF11chromatography, and three of them were selected for sequence determination. The viruses were named as HetRV10-an1, HetRV7-an1, and HetRV3-an2. Based on their genome sequences, new primers were designed to allow screening for the presence of similar viruses in the remaining Heterobasidion isolates using RT-PCR (reverse transcriptase- polymerase chain reaction). Using this method, HetRV10-an1, HetRV7-an1, and HetRV3-an2 were found from 4, 3 and 3 Heterobasidion isolates. All the viruses found were related to previously described members of Partitiviridae. The RdRp (RNA-dependent RNA polymerase) fragment of HetRV7-an1 was 2297bp long and it showed 70% similarity to HetRV2-pa1 from H. parviporum (GenBank accession HM565953). The RdRp segment of HetRV3-an2 virus was 1897 bp long and 65% similar to HetRV3-ec1 from H. ecrustosum (FJ816271). The 871 bp partial sequence of HetRV10-an1 was somewhat similar (58%) to the Vicia faba partitivirus 1 (DQ910762), and can be considered a new partitivirus species. HetRV7-an1 and HetRV3-an2 virus were found from three and two different H. annosum genets, respectively, in the study plot, while HetRV10-an1 occurred in only one genet (all four H. annosum isolates representing the same genet). In conclusion, H.annosum of the studied forest plot hosts a diverse pool of mycoviruses.

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L22 Engineering resistance to the plant pathogen Phytophthora infestans by manipulation of phytoalexin pathways. Artemis Giannakopolou12, Schornack, S.1, Kamoun, S.1, O’Maille, P.2

1The Sainsbury Laboratory, Norwich, NR4 7UH, UK; 2John Innes Centre, Norwich, NR4 7UH, UK The production of phytoalexins in plants is part of their long-term resistance, the so-called systemic acquired resistance (SAR). Phytoalexins are low molecular mass antimicrobial secondary metabolites, absent in healthy tissues and are synthesised de novo after stress. We investigate the role of the phytoalexin capsidiol sensitivity/resistance as a determinant of host-specificity in Phytophthora-Solanaceae interactions. The fungus-like oomycete Phytophthora infestans, causal agent of the late blight in potato and tomato is unable to successfully colonize capsidiol-producing plants such as pepper and tobacco. We confirmed that P. infestans growth can be inhibited by low capsidiol concentrations while inhibition of pepper-infecting P. capsici requires much higher capsidiol levels. We cloned genetic elements of capsidiol biosynthesis, a sesquiterpene synthase (TPS) and a cytochrome p450/reductase and plan to identify the role of capsidiol in the in P. infestans-host system by silencing and overexpression. We also started to evaluate the sensitivity of Phytophthora strains and species to capsidiol and plan to extend this to alternative phytoalexin repertoires generated by mutants of the tobacco TPS gene. To clarify the mode of action of capsidiol in arresting the growth of P. infestans, a metabolomics study will be launched. To screen the sensitivity of various Phytophthora stains in potato’s chemical defence compounds, a reprogramming of Nicotiana benthamiana through expression of TPS/ p450 genes is scheduled. We currently set up gas chromatography coupled with mass spectrometry to quantify capsidiol production over the time-course of experiments. On the long term we aim to engineer tomato and potato plants for production of capsidiol, through reconstitution of the capsidiol biosynthetic pathway from tobacco into potato and tomato in order to evaluate crop resistance versus a panel of Phytophthora strains.

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L23 Engineering pathogen resistance in crop plants David B. Collinge Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871-Frederiksberg C, Denmark Disease resistance is undoubtedly the most effective means for controlling plant diseases – when available. Unfortunately, effective natural sources of disease resistance are rarely available for necrotrophic pathogens. Soil-borne pathogens are often necrotrophic and it is therefore often difficult to combat the diseases they cause using resistance. Can transgenic disease resistance offer attractive approaches for controlling these difficult diseases? As far as I am aware, there are currently no success stories where transgenic disease resistance against fungal pathogens has been introduced into the field, let alone against soil-borne diseases. Is this entirely due to the caution of authorities given the negative public opinion that encompass transgenic technologies or is this because no effective solutions exist yet? Which biological approaches are the most promising? Are there prospects that they can provide effective control against soil-borne diseases. I will present the issues and discuss the biological prospects for transgenic disease control. In our own research, we are studying the mechanisms used by cereals to perceive and react against pathogen attack. Our efforts are concentrated on the NAC and CRK gene families of cereals which are transcription factors and cysteine-rich receptor-like protein kinases, respectively. Characterisation of specific members of these gene families has demonstrate that suppression of HvNAC6 but not HvNAC1 or HvNAC4 expression by RNAi results in increased susceptibility to Blumeria graminis. In contrast, RNAi studies with HvCRK1 resulted in increased resistance to Blumeria. These studies are being extended to other members of the gene families as well as to other pathogens of cereals. 1. Chen,Y.J., Lyngkjaer,M.F., and Collinge,D.B. 2012. Future prospects for genetically

engineering disease resistance plants. In: G.Sessa (Ed.), Molecular Plant Immunity, John Wiley & Sons, New York.

2. Collinge,D.B., Jørgensen,H.J.L., Lund,O.S., and Lyngkjær,M.F. 2010. Engineering pathogen resistance in crop plants - current trends and future prospects. Annu Rev Phytopathol 48: 269-291.

3. Collinge,D.B., Lund,O.S., and Thordal-Christensen,H. 2008. What are the prospects for genetically engineered, disease resistant plants? Eur J Plant Pathol 121: 217-231.

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Poster Abstracts

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List of posters P1 Gene expression analysis of perennial rye grass during pink snow mould infection M. Abdelhalim, Å.Ergon, M.R.Kovi, A.Kukapareddy, M.B.Brurberg, I.S.Hofgaard, A.M.Tronsmo and O.A.Rognli P2 Fungal effectors shaping extrahaustorial membrane formation: the cellular origin and molecular basis of extrahaustorial membrane formation Geziel Aguilar, Mark Kwaaitaal, Carsten Pedersen, Hans Thordal-Christensen.

P3 Validation of barley powdery mildew effector candidates and identification of their barley targets Ali Ahmed, Carsten Pedersen and Hans Thordal-Christensen P4 Molecular studies of Rhizoctonia solani and its corresponding resistance genes in sugar beet Louise Andersson, Mark Varrelmann, Thomas Kraft, Britt-Louise Lennefors, Christina Dixelius

P5 Identification and Characterization of LysM-NKs in Lotus japonicus Mai-Britt Brøndum, Jens Stougaard, Simona Radutoiu P6 Identification of viruses infecting potatoes (Solanum tuberosum) in Mbeya region, Tanzania. Evangelista Chiunga and Jari P. T. Valkonen

P7 Biological Control of Soft Rot of Potato Tubers under Storage Conditions Hadizadeh, I., Pirhonen, M. P8 Host-pathogen interactions in closely related Phytophthora species Hosseini, S., Heyman, F., Funck Jensen, D., and Karlsson, M. P9 The mechanism of plant quantitative resistance in wheat Bulat Islamov, Hilma Peusha, Irena Jakobson, Kadri Järve

P10 Construction of mycotoxin-induced cDNA libraries of Clonostachys rosea IK726 based on Suppression Substractive Hybridization Chatchai Kosawang, Magnus Karlsson, David Collinge and Dan Funck Jensen P11 Establishing and screening a Lotus japonicus LORE1 mutant population Anna Malolepszy, Dorian Urbanski, Jens Stougaard, Stig Uggerhøj Andersen P12 Molecular identificacion of seed-borne fungi affecting bean seeds (Phaseolus vulgaris L. cv. INTA Rojo) in Nicaragua Marcenaro. D. I and, Valkonen J.P.T P13 Fungal pathogens infecting the cultivated Sunagoke moss hamper sustainable greening in urban environments and also cause diseases in field and horticultural crop plants Eeva M. Marttinen, Mikko T. Lehtonen, Motomu Akita & Jari P.T. Valkonen

P14 Transcriptome profiling of potato tubers infected by soft rot bacteria in different storage conditions Peivastegan, B., Pirhonen, M. P15 Population genetics and epidemiology of Puccinia striiformis on cereals and grasses: Host boundaries and off-season survival Tine Thach, Annemarie Fejer Justesen, Mogens Støvring Hovmøller

P16 Development of Taphrina Species into Genetically Tractable Model Pathogens Brook Tekele, Timo Sipilä, Ajay Anand, Shaman Narayanasamy,Kirti Prakash, Jarkko Salojävi, Lars Paulin, Petri Auvinen and Kirk Overmyer

P17 Helper component proteinase of Potato virus Y as a virulence determinant in plants Yanping Tian and Jari P.T. Valkonen P18 Viral RNase III as a silencing suppressor Isabel Weinheimer, Minna-Liisa Rajamäki and Jari Valkonen. P19 Epidemiology and management of coconut lethal yellowing disease in central region of Mozambique João Bila, Ana Mondjana, Berit Samils and Nils Högberg

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P1 Gene expression analysis of perennial rye grass during pink snow mould infection M. Abdelhalim1, Å.Ergon1, M.R.Kovi1, A.Kukapareddy1, M.B.Brurberg1, I.S.Hofgaard2, A.M.Tronsmo1 and O.A.Rognli1 1Dept. of Plant and Environmental Sciences, Norwegian University of Life Sciences, P.O. Box 5003, NO 1432 Aas, Norway, 2 Bioforsk Plant Health and Plant Protection. Pink snow mould caused by Microdochium nivale (Fr.) Samuels & Hallett is a serious disease in temperate grasses in cold to temperate regions of the northern hemisphere (Tronsmo, 2001). Resistance to snow mould is induced by cold acclimation, and most previous studies on snow mould resistance have been performed on cold acclimated plants. Cold acclimation induces general pathogen defense responses especially to low-temperature fungi. We aim to understand defense mechanisms that are independent of cold acclimation and possibly more specific to pink snow mould. Understanding the nature of resistance to snow mould independent of cold acclimation will help to improve and develop resistance varieties especially for the predicted future climate change. We will start with screening ten genotypes from four different varieties of perennial ryegrass for cold acclimation-independent pink snow mould resistance. Afterwards we will select the most resistant and susceptible genotypes within one population for transcriptome analysis during infection by the use of RNA-Seq technology. This will allow us to obtain important information about how plants respond to snow mould infection without the effect of cold acclimation. References Tronsmo, A.M., Hsiang, T., Okuyama, H., Nakajima, T. 2001. Low temperature diseases caused by Microdochium nivale 75-86 in: Iriki, N., Gaudet, D.A., Tronsmo, A.M., Matsumoto, N., Yoshida, M., Nishimune, A., (eds), Low Temperature Plant Microbe Interactions Under Snow, Japan, Hokkaido National Agricultural Experiment Station.

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P2 Fungal effectors shaping extrahaustorial membrane formation: the cellular origin and molecular basis of extrahaustorial membrane formation Geziel Aguilar1, Mark Kwaaitaal, Carsten Pedersen, Hans Thordal-Christensen.

1 Defence Genetics, Dep. of Agriculture and Ecology, Faculty of Life Sciences, Copenhagen University. Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew disease in barley (Hordeum vulgare L.), is a biotrophic pathogen that establishes an intimate intracellular relation with its host. The pathogen requires an interface with the host to exchange nutrients and signalling molecules. For this purpose the fungus makes a haustorium, which is a feeding structure that enters the host cell and during infection a host-derived extrahaustorial membrane (EHM) forms around it. The EHM is the referred host-pathogen interface which is crucial for fungal establishment. Formation of EHM likely promotes fungal-induced changes in the plant cell’s endomembrane trafficking. Our present research focuses on understanding how the barley powdery mildew induces formation of the EHM. Vesicle transport in the host cell is regulated by various proteins (e.g. small GTPases and SNARE complexes), which comprise potential targets for fungal effectors. To identify such effectors and their targets we use a Bgh cDNA library ectopically expressed in yeast or Arabidopsis to screen for disrupted secretion phenotypes. The library is made from Bgh mRNA extracted at a critical time point during infection, when the EHM is being formed. Additionally, Transient Induced Gene Silencing (TIGS) of proteins required for particular steps in endomembrane trafficking allows the assessment of the role of specific vesicle trafficking pathways in disease susceptibility or resistance. Identification of host components facilitating infection and characterization of fungal effectors targeting the host cellular machinery will bring insight into the host origin of the EHM and the molecular processes governing the establishment of the Bgh intracellular interface with the host. This knowledge opens opportunities for the development of bioengineering strategies to improve plant resistance against powdery mildews and pathogens with a similar lifestyle.

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P3 Validation of barley powdery mildew effector candidates and identification of their barley targets Ali Ahmed, Carsten Pedersen and Hans Thordal-Christensen Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Denmark

Barley powdery mildew (Blumeria graminis f. sp. hordei) and barley interaction is used as a model system to understand plant–microbe interaction. Barley powdery mildew is a biotrophic fungus, forming specialized feeding structure “haustoria” inside the cell and develops intimate relationship with host cell. Previous studies identified more than 400 effector candidates which are believed to be delivered to plant cell and suppress barley defence responses and/or facilitate susceptibility (Bindschedler et al., 2009, Godfrey et al., 2009 and 2010). Most effector candidates share N-terminal Y/F/WxC-motif downstream of signal peptides which possibly required in transferring effectors from haustoria to plant cell. In this study, we aim to confirm the function of some effector candidates and identify their barley targets. Currently, Host Induced gene Silencing (HIGS) is being used to validate some effector candidates (Nowara et al., 2010). Over expression study of effector candidates will be carried out in single cell transformation using biolistic bombardment (Douchkov et al., 2005). Yeast two hybrid approach will be used to identify barley targets for candidates showing effector function. The function of the effector target proteins will be studied using Transient induced gene silencing (TIGS) by RNAi and over expression in barley epidermal cells. Barley target/effector interactions will be studied in depth using localization study, BIFIC, and others approaches.

References: Bindschedler et al., 2009. In Planta Proteomics and Proteogenomics of the Biotrophic Barley Fungal Pathogen Blumeria graminis f. sp. Hordei. Molecular & Cellular Proteomics 8.10. Godfrey et al., 2009. A proteomics study of barley powdery mildew haustoria. Proteomics , 9, 3222–3232 Godfrey et al., 2010. Powdery mildew fungal effector candidates share N-terminal Y/F/WxC-motif. BMC Genomics, 11:317 Douchkov et al., 2004. A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells. Molecular Plant-Microbe Interactions 18 (8):755-761 Nowara et al., 2010. HIGS: Host-Induced Gene Silencing in the Obligate Biotrophic Fungal Pathogen Blumeria graminis. The Plant Cell, 22: 3130–3141.

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P4 Molecular studies of Rhizoctonia solani and its corresponding resistance genes in sugar beet Louise Andersson1, Mark Varrelmann2, Thomas Kraft1, Britt-Louise Lennefors1, Christina Dixelius3

1 Syngenta Seeds, Säbyholmsvägen 24, Landskrona, Sweden; 2Institute of Sugar Beet Research (IfZ), Holtenser Landstr. 77, Göttingen, Germany; 3Uppsala BioCenter, Dept. of Plant Biology and Forest Genetics, SLU, Box 7080, Uppsala, Sweden Rhizoctonia solani is a soil-born fungal pathogen with a wide host range. R. solani is subdivided into anastomosis groups (AGs) based on hyphal fusion between compatible strains. AG2 and AG4 are reported to cause most disease problems in sugar beets. R. solani is a problem in all areas where sugar beets are grown. In USA R. solani is one of the most serious root diseases in sugar beets with great yield losses. It can occur in spots in a field or entire fields can be destroyed. The disease could partly be controlled with fungicides, but the timing is difficult and the best way to control the disease is to grow resistant varieties. The resistance in sugar beets to R. solani is quantitative and there is often a small decrease in yield linked to the resistance trait. If the resistance genes underlying the QTLs could be identified, then new molecular markers could be designed and used to improve the selection efficiency in breeding. I just started my PhD studies and my work is on R. solani and its interaction with sugar beet. The work will be divided in two parts; the fungi and the sugar beet. Deep sequencing of R. solani AG2-2IIIB transcriptome will be done. With bioinformatic tools pathogenicity factors in the fungi will be identified. This information will be compared to AGs with other host ranges. In order to confirm the bioinformatic information a high throughput transformation system for R. solani will be established. We need to be able to complement genes or knock-down genes of interest for more functional studies. The other part is deep sequencing of the sugar beet transcriptome, comparing resistant and susceptible genotypes under infection with R. solani. Here we have a sugar beet reference genome to compare with and we will try to identify resistance genes to R. solani. Expression analysis with qRT-PCR on candidate genes identified in the transcriptome sequencing will be performed among other analysis.

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P5 Identification and Characterization of LysM-NKs in Lotus japonicus Mai-Britt Brøndum1, Jens Stougaard1, Simona Radutoiu1 1 Centre for Carbohydrate Recognition and Signalling, Department of Genetics and Molecular Biology, Aarhus University, Denmark The LysM motif is a 45-65 aa protein domain, identified in both prokaryotes and eukaryotes, which has been shown to bind different carbohydrate structures such as the peptidoglycan (PGN) of bacterial cell walls or chitin and chitooligomers derived from fungal cell walls. In plants LysM-RLKs have been shown to be part of signaling cascades involved in the recognition of bacterial and fungal microbes. In the legume Lotus japonicus the LysM-RLKs NFR1 and NFR5 have been shown to be involved in the recognition of symbiotic nitrogen fixing rhizobia, and in rice the LysM-RLK CERK1 and the LysM-Non Kinase(NK) CeBIP have shown to be essential for recognition and defense signaling against fungal pathogens. Recently, two other LysM-NKs, LyM1 and LyM3, have shown to be essential for the recognition of bacterial pathogens in Arabidopsis. Thus, not only LysM-RLKs, but also LysM-NKs are emerging as important players in the recognition of microbes in plants, and more research is needed to shed light on the evolution of this gene family and its potential roles in plant-microbe interactions. The aim of this study is to identify and characterize the LysM-NK genes in Lotus japonicus. The LysM-NKs potential role in the recognition of bacterial and fungal microbes will be evaluated by detailed characterization of transcript regulation in response to treatment with different microbe derived molecules and in response to inoculation with symbiotic rhizobia and mycorrhiza. Also the phenotype of selected LysM-NK mutants, obtained from the LORE1 collection, will be evaluated.

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P6 Identification of viruses infecting potatoes (Solanum tuberosum) in Mbeya region, Tanzania. Evangelista Chiunga1, 2and Jari P. T. Valkonen1

1Department of Agricultural Sciences, P.O. Box 27, FI-00014 University of Helsinki, FINLAND, 2 Uyole Agricultural Research Institute, P.O. Box 400, Mbeya, TANZANIA The potato (Solanum tuberosum) is an important food and cash crop in Tanzania, which was introduced in the early 1920s and is gaining importance both at the farm level and in urban markets. Despite the importance of the potato as both cash and food crop in Tanzania, the low yield has been a major problem in its production because of insufficient use of improved agricultural technologies being used by potato farmers in the country. Moreover, virus diseases have a major contribution to lower potato yields in developing countries such as Tanzania due to their warmer and more humid climates. Worldwide, Potato virus Y (PVY), Potato virus X (PVX), Potato virus A (PVA), Potato leafroll virus (PLRV), Potato virus M (PVM) and Potato virus S (PVS) are considered the most important in terms of their distribution and effect on the yield. No information is currently available on the occurrence of potato viruses in Tanzania. A brief survey in the southern highlands of Tanzania (Mbeya region) revealed diverse virus-like symptoms in different potato fields such as yellowing mosaic, leaf rolling, stunted plants, yellow spots and curled leaves, as well as black veins on the underside of the rolled leaves. Double antibody-sandwich (DAS) ELISA was applied for the detection of PVM, PVY, PVA, PVS, PVX and PLRV, and all six viruses were detected in the sampled leaves. PVS was the most prevalent virus, while PVY and PVM were the least prevalent viruses. Three infections (PLRV, PVA and PVX) were confirmed by RT-PCR by using leaf samples stored on FTA® cards. In ongoing research, RT-PCR analysis for PVS, PVM and PVY followed by phylogenetic analysis using the sequences of coat protein (CP)-encoding region will be performed.

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P7 Biological Control of Soft Rot of Potato Tubers under Storage Conditions Hadizadeh, I., Pirhonen, M. Department of Agricultural Science, P.O. Box 27, 00014 University of Helsinki Post harvest potato soft rot caused by pectinolytic Pectobacterium and Dickeya species is a bacterial disease creating economic damage in potato production. Effective management to control soft rot in tubers stored is largely absent. There are no chemical control measures available nor are there any resistant varieties. Possibilities to use biological control agents (BCAs) for control of Dickeya solani and Pectobacterium caratavorum were investigated under storage condition. A collection of antagonists were isolated from rotten potato mass and identified by16S rDNA based PCR technique and in addition, 54 isolates from different bacteria genera were chosen as candidate antagonists from different countries. Four isolates revealed high antagonistic activity on dual agar method in vitro. To analyse the effect of the antagonists in storage condition, whole potato tubers were inoculated with combinations of the pathogens and the antagonist isolates strongly inhibited soft rot at 55.6% - 89% and 66.6% - 100% ratio by D. solani and Pectobacterium caratavorum, respectively. Their ability to colonization of potato surface was investigated with antibiotic resistance mutants. Four isolates showing sufficient performance based on colonization /growth ability on potato peel were selected for evaluating the population dynamic of the pathogen and the influence of antagonist treatment on development of soft rot over time, temperature and wounded or unwounded tubers in storage condition. Two antagonists provided a good level of survival. The best BCAs upon reduction of the percent weight loss, the surface area with rot and the number of wound with rot development were selected for further studies. The next objective is to explore transcriptomic profiling of BCAs in its early interactions with potato tuber using Solexa transcriptome analyses for revealing/ discovering new important genes for biocontrol traits in the antagonistic strains.

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P8 Host-pathogen interactions in closely related Phytophthora species Hosseini, S., Heyman, F., Funck Jensen, D., and Karlsson, M. Uppsala BioCenter, Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Box 7026, SE-75007, Uppsala, Sweden. The genus Phytophthora (phylum Oomycota) contains destructive plant pathogens. The phylogenetic tree of the genus shows that closely related species can have very different biology in this respect. Experimental data on oomycete pathogenicity factors play a key role towards better understanding of oomycete-plant interactions. The putatively novel species P. pisi (Heyman et al. in prep) causes root rot in pea, and is an emerging disease in southern Sweden. The most closely related species is P. sojae. The aim of this study is to gain an understanding of the mechanisms underlying the differentiation of a subgroup of root-infecting Phytophthora species and the genetic and biochemical basis for host species specificity. Thus, one part of the project aim to determine the host spectrum of closely related Phytophthora species and the correlation with zoospore root attraction. In addition, we aim to analyse transcriptomes and proteomes of P. pisi during infection-related conditions. Moreover, the interaction of P. pisi effectors with their target proteins in pea will be tested in an in vivo 2-hybrid system. Project 1: Host spectrum determination of closely related Phytophthora species and correlation with zoospore-root and zoospore-isoflavones attraction assays: P. sojae, P. niederhauseri, P. pisi and P. vignae are closely related species with different host spectra. In the project, the pathogenicity of these species will be tested against a selection of legumes, using inoculated pot experiments. Zoospores of root-infecting oomycetes have chemotactic and electrotactic abilities. Zoospore isoflavones assay showed that the closely related root-infecting Phytophthora species have differential attraction patterns towards a range of isoflavones. The data suggests that chemotaxis of zoospores to isoflavones could be involved in recognition of the host and for initiating infection

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P9 The mechanism of plant quantitative resistance in wheat Bulat Islamov1, Hilma Peusha2, Irena Jakobson3, Kadri Järve4

1,2,3,4 Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia Wheat powdery mildew Blumeria graminis DC. f. sp. tritici is widespread pathogen in regions with humid temperate climate. Several types of resistance have been described to date. Race nonspecific resistance that is controlled by genes with intermediate effects is an alternative mechanism to gene-for-gene interaction and can be used to reduce powdery mildew infestation in the field. The purpose of this study is to reveal mechanisms of disease resistance in interaction of wheat and powdery mildew pathogen. Interactions between plant host and fungal pathogens are less frequently analysed in wheat than in Arabidopsis and barley and therefore less understood. Previous research of our group resulted in mapping and identification of QTL conferring durable resistance to powdery mildew transferred to bread wheat Triticum aestivum L. from tetraploid wheat Triticum militinae Zhuk. et Migusch. Because of large size and complexity of wheat genome positional cloning is impossible. The locus carrying the main QTL was transferred to telosomic chromosome. Then telosomic chromosomes carrying resistance gene(s) were isolated by flow sorting and chromosome arm-specific BAC library has been created (collaboration with Dr. Jaroslav Dolezel lab, Czech Republic). The BAC library needs to be screened with markers linked to the trait of interest. Current task is to find differences in host cell response of distinct genotypes of introgressive wheat lines to powdery mildew infection and to find cytological effects of different QTL.

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P10 Construction of mycotoxin-induced cDNA libraries of Clonostachys rosea IK726 based on Suppression Substractive Hybridization Chatchai Kosawang1,2, Magnus Karlsson2, David Collinge1 and Dan Funck Jensen2 1 Department of Plant Biology and Biotechnology, University of Copenhagen, Denmark; 2 Department of Forest Mycology and Plant Pathology, SLU, Sweden. Clonostachys rosea IK726 is a mycoparasitic fungus effective in controlling several plant diseases. Its successes include Fusarium culmorum that produces a non-steroidal oestrogenic mycotoxin Zearalenone (ZEN) that inhibits fungal growth and Deoxynivalenol (DON) that is a strong protein inhibitor. C. rosea can detoxify ZEN through the enzyme Zearalenone lactonohydrolase, which cleaves ZEN into a less toxic metabolite. Nonetheless, it is not known whether additional mechanisms to detoxify or tolerate ZEN and DON are acting in C. rosea. In this study suppression subtractive hybridization (SSH)-based cDNA libraries of ZEN- and DON-induced genes are being generated in an attempt to identify genes that are expressed to cope with DON and ZEN. Thus far the DON library was developed with 912 clones generated while the ZEN library is ongoing. Our results obtained from approximately 480 clones from the DON library unveiled transcripts encoded proteins involved with stress responses, cellular transport and metabolism. These selected transcripts include plasma membrane ATPase, cytochrome P450 protein family, cytochrome C oxidase and ATP-binding proteins. We hope that results of this study will enhance our knowledge of the processes underlying detoxification of mycotoxins as well as antagonistic interaction of mycoparasitic fungi in general.

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P11 Establishing and screening a Lotus japonicus LORE1 mutant population Anna Malolepszy, Dorian Urbanski, Jens Stougaard, Stig Uggerhøj Andersen Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology, University of Aarhus, 8000 Aarhus C, Denmark. Lotus japonicus is a model legume plant, which is able to fix atmospheric nitrogen by symbiosis with rhizobia. Up to now, a number of genes involved in symbiosis were discovered. To facilitate this field, we developed new, reverse genetic tool for insertional mutagenesis - LORE1 resource. Lotus retrotransposon 1 (LORE1) belongs to Gypsy-type retrotransposon and is 5kbp long. LORE1 can be derepressed during tissue culture, which leads to a few new insertions in next generations. It was shown that LORE1 inserts randomly, without sequence preferences. A two-dimensional barcoding and pooling strategy, as well as LORE1 border fragment amplification is used to simultaneously identify novel insertions in 9,000 individual plants in one Illumina run. Thanks to that, we hope to find novel components in the signal transduction pathway during establishment of plant-bacteria symbiosis. Our main focus is the newly discovered fix- mutant. The nodule organogenesis seems to be impaired in these plants. Instead of the fully developed nodules, only black bumps are observed on the mutant roots. We wish to investigate what causes the disruption of the process: is it due to the defense response or due to other factors like impaired cell growth and differentiation. We would like to compare the transcription level of defense-related genes in this mutant and the wild type.

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P12 Molecular identificacion of seed-borne fungi affecting bean seeds (Phaseolus vulgaris L. cv. INTA Rojo) in Nicaragua Marcenaro. D. Iª b and, Valkonen J.P.T a† ª Department of Agriculture Sciences, University of Helsinki, P.O. Box 27, FIN-00014, Finland; b Nicaraguan Institute of Agricultural and Technology (INTA), National Programme of Seed and Biotechnology, Nicaragua. Common bean (Phaseolus vulgaris L.) is an important legume crop grown widely around the world. However, production and consumption of common beans have decreased because of lack of cultivars yielding well under the local conditions, inappropriate agricultural practices, and problems with diseases and pests. Seed-borne fungi are thought to represent a huge limitation to bean production in Nicaragua and cause large yield losses. However, identification of the fungal seedborne pathogens in common beans has gained little attention. The present study aimed to identify seedborne fungal pathogens in a new important bean variety, INTA Rojo, in Nicaragua. Fungi were isolated from 6 bean lots of cv. INTA Rojo which represent the main production areas of INTA Rojo in Nicaragua. Fungi were grown out from surface-sterilized beans. Pathogenicity of 133 fungal isolates was tested by inoculation of bean seedlings grown from pathogen-free seed. Eighty-seven isolates caused symptoms of varying severity including discoloration, necrotic lesions, rottening or death of bean seedlings. Pathogenic isolates were placed to eight phenotypically distinguishable groups (phenogroups) based on cultural characteristics and further identified by analysis of internal transcribed spacer (ITS1 and ITS2) sequences of the ribosomal genes. PCR products were purified and sequenced. The isolates belonged to the following species or genera, as judged based the highest ITS sequence identities identified: Fusarium chlamydosporum, F. equiseti and F. incarnatum (phenogroup 1); Macrophomina phaseolina (phenogroup 2); both phenogroups were found in all regions. Lasiodiplodia theobromae (teleomorph Botrysphaeria rhodina) (phenogroup 3) was frequent in Boaco and Carazo. Corynespora cassiicola, (phenogroup 4) was found in Carazo. Collectotrichum gloesporiodes (teleomorph Glomerella cingulata) and C. capsici (phenogroup 5) were most common in Boaco and Matagalpa. Penicillium citrinum (phenogroup 6) were found Estelí, Carazo and Matagalpa. Aspergillus flavus (phenogroup 7); were frequent in Matagalpa and Estelí. Diaporthe sp. (phenogroup 8). These results revealed many seedborne fungal pathogens of common beans for the first time in Nicaragua. Keywords: Seed-borne fungi, Phaseolus vulgaris, Fusarium chlamydosporum, Macrophomina phaseolina,

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P13 Fungal pathogens infecting the cultivated Sunagoke moss hamper sustainable greening in urban environments and also cause diseases in field and horticultural crop plants Eeva M. Marttinen1, Mikko T. Lehtonen1, Motomu Akita2 & Jari P.T. Valkonen1

1Department of Agricultural Sciences, PO Box 27, FI–00014 University of Helsinki, Finland 2Department of Biotechnological Sciences, Kinki University, Kinokawa, Wakayama, 649-6493, Japan Note: EMM and MTL contributed equally. Drought and heat tolerance of the Sunagoke moss (Racomitrium japonicum) and the low thermal conductivity of the dry moss tissue offer novel greening and insulation possibilities of roofs and walls to mitigate heat island phenomenon in urban environments. However, damage may appear in the moss panels (Akita et al. 2011, Sci. Total Environ. 409: 3166-3173). In this study we characterized fungi associated with the damaged areas of the Sunagoke moss panels. Fungi were identified and tested for pathogenicity on R. japonicum, Physcomitrella patens and cultivated vascular plants under controlled conditions. Alternaria alternata, Fusarium avenaceum and Fusarium oxysporum caused severe necrosis and death, whereas Cladosporium oxysporum and Epicoccum nigrum caused milder discoloration or chlorosis in both moss species. The F. avenaceum isolate caused also root and crown rot in barley and reduced germination of tomato and carrot. C. oxysporum caused necrosis on carrot seedlings after emergence. This study demonstrates that fungi can cause economically significant diseases in moss by damaging the commercially used moss panels. Results also showed that the natural host range of some plant-pathogenic fungi is wider than known, covering bryophytes and vascular plants. The moss panels may be contaminated with fungal pathogens, disseminated from cultivated vascular plants, and may also act as reservoirs of fungal pathogens of vascular plants.

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P14 Transcriptome profiling of potato tubers infected by soft rot bacteria in different storage conditions Peivastegan, B., Pirhonen, M. Department of Agricultural Sciences, P.O. Box 27, 00014 University of Helsinki Potato soft rot is one of the destructive postharvest diseases caused by pectinolytic Pectobacterium and Dickeya species (previously called Erwinia). The bacteria can be present in tubers as latent infection for a long time, and the start of an active infection has been associated with wet conditions in the storage rooms. The pathogen can enters the tubers from wounds or natural lenticels in tubers or alternatively, it spreads into the tuber via vascular system during the tuber formation. Recognition of pathogen leads to a complex signaling cascade leading to inducible defense responses in potato. These responses include cell wall strengthening, oxidative burst, metabolic changes and the expression of a large amount of defense related genes. Abiotic stress, such as high humidity and depletion of oxygen, affect the defense responses and lead to rotting of the tubers by pathogen. The effect of abiotic stresses, especially high humidity as a most important factor affecting rotting of potato during storage, has previously never been modeled using modern methods such as transcription analysis. Recently, complete genome sequence information from potato has yielded the ability to perform high throughput, genome wide screens of gene activity and has boosted application of a range of a new technologies to study plant gene functions during host - pathogen interactions. My study focuses on comparison of the gene expression in the potato tuber and soft rot bacteria during interaction to identify changes that proceed rotting. Application of new sequencing technologies, Illumina/solexa deep sequencing allows us to simultaneously sequence millions of different DNA molecules and develop a large dataset corresponding to transcribed genes in the pathogen and host. We will utilize this technology on RNA samples derived from potato tubers infected with Pectobacterium/Dickeya, both inoculated tuber and noninoculated controls stored either in diseases reducing condition (air circulation and low humidity) or in disease inducing condition (closed container with high humidity and low oxygen level).

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P15 Population genetics and epidemiology of Puccinia striiformis on cereals and grasses: Host boundaries and off-season survival Tine Thach1, Annemarie Fejer Justesen1, Mogens Støvring Hovmøller1

1 Aarhus University, Dept. of Agroecology, Forsøgsvej 1, DK-4200 Slagelse, Denmark The yellow rust disease caused by Puccinia striiformis sensu lato is an obligate biotrophic fungal pathogen infecting cereals and cultivated grasses. The spores are air-borne and can spread over long distances between regions and continents. Currently, yellow rust is causing the most damaging disease epidemics on wheat, and causing crop losses up to 25%. In recent years yellow rust have changed and adapted to higher temperature regimes allowing epidemic development in warmer areas along with increased pathogen aggressiveness. A new highly aggressive race of P. striiformis was sampled from triticale and widespread in 2008 and 2009 in Scandinavia and, in fact, able to infect cultivars of different cereals. The aim of the project is to investigate the geographical and evolutionary relationship between P. striiformis infecting cereal crop species, their wild relatives and grasses from distinct epidemiological areas. Yellow rust isolates from the historic “Stubbs collection” and isolates from different epidemiological zones and at different times will be investigated by appropriate DNA markers, and virulence assays where possible. The results should allow testing hypotheses about the role of local off-season pathogen survival. Furthermore, P. striiformis host boundaries and the underlying host resistance genetics will be investigated via cross-infection studies using P. striiformis from different cereals, wild relatives of these, and possibly some grasses. The phenotypic response of a successful or non-successful infection will be investigated by both macroscopic and microscopic techniques. Preliminary results from my PhD project, which started by February 1st 2012, will be presented at the course.

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P16 Development of Taphrina Species into Genetically Tractable Model Pathogens Brook Tekele1, Timo Sipilä1, Ajay Anand1, Shaman Narayanasamy1,Kirti Prakash1, Jarkko Salojävi1, Lars Paulin2, Petri Auvinen2 and Kirk Overmyer1

1Plant Biology, Department of Biosciences, University of Helsinki, 00014 Helsinki, Finland. 2Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland. Introduction: Taphrina is a fungal genus in the phylum Ascomycota which comprises nearly 100 species (Rodrigues and Fonseca, 2003). All known species of the genus Taphrina are plant parasites (Mix, 1969). Members of the genus Taphrina display dimorphic life cycle switching between a saprobic unicellular yeast stage and a parasitic multicellular fungal/filamentous stage upon finding a suitable host (Nadal et al., 2008). The yeast stage exists as a haploid and the mycelial stage is dikaryotic/multinucleated. The yeast stage can be grown on a culture media but the fungal stage is an obligate parasite (Mix, 1969). Why to study Taphrina? Agricultural interest: Taphrina infect many economically important wild and cultivated trees like Birch and Prunus trees (peach, plum and cherry) (Agrios, 2005). Medical interest: the genus Taphrina is closely related to the human lung pathogen Pneumocystis. Biological interest: the evolutionary line of Taphrina represents early divergence in the higher fungi (Dikarya). Objectives: Little molecular work has been done on Taphrina and this project aims at filling some of this research gap. The general objective of this project is to develop Taphrina species into genetically tractable model pathogens and specific objective includes generating mutant Taphrina yeast strains using gene knockout and/or over-expression strategies for genes of interest that putatively promote virulence in Taphrina spp.

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P17 Helper component proteinase of Potato virus Y as a virulence determinant in plants Yanping Tian1 and Jari P.T. Valkonen1

1Department of Agricultural Sciences, University of Helsinki, FI-00014, Helsinki, Finland Potato virus Y (PVY), the type member of genus Potyvirus, is one of the most common and destructive viruses infecting potato and other crops of Solanaceae such as pepper, tobacco and tomato. Resistance genes that control PVY have been identified in cultivated and wild potato species and have been used in potato breeding for many years (Lana and Benson 1967; Schenk 1989; Jones 1990; Brandolini, Caligari et al. 1992; Saezvasquez, Theoduloz et al. 1993; Flis 1995; Schaad, Lellis et al. 1997). Various strain groups have been described, including PVYO, PVYN, PVYC and PVYZ, as defined based on potato resistance genes for hypersensitive response (HR) that each specifically detect only strains of one of these strain groups (Singh et al., 2008). Recent studies showed that PVYC Hcpro is the effectors corresponding to the Nc gene (Moury, Caromel et al. 2011) (Ref), However, no detail information were available for the PVYO strain. In this study, we showed that HCpro of PVYO can elicit the HR on the hosts carrying Ny gene. Furthermore, we mapped the central domain of PVYO HCpro is essential for induction of HR. It located at the hinge region of HCpro between two helix domains. It contained several important motifs including IGN, CC(S)C, PTK. Many functions have mapped to this region in previous study, e.g., amplifications, post transcriptional gene silencing (PTGS), synergistic, RNA binding domain B, coat protein binding motif, Ribonucleoprotein, movement, RNA recognition motif, self-interaction and inhibiting Hua enhancer 1 methyltransferase activity. Together with structure previous reported and predicted in this study, lead to the hypothesis that region a contains active sites needed for various function, and that the hinge domain regulated their accessibility by moving domain c+d to mask or expose domain a. the movement of the hinge domain could, in turn be regulated by interaction with various host or viral partners (Plisson, Drucker et al. 2003).

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P18 Viral RNase III as a silencing suppressor Isabel Weinheimer, Minna-Liisa Rajamäki and Jari Valkonen. Department of Agricultural Sciences, University of Helsinki, Finland, 00014. RNA silencing (RNAi) is a major antiviral mechanism in plants, initiated by double-stranded RNA (dsRNA). DsRNA is cleaved by RNase III enzymes of Classes 2 and 3 into ~20-25 nt small RNA (siRNA), which are primary actors in RNAi. We have recently identified an RNase III-like enzyme encoded by the RNA genome of sweetpotato chlorotic stunt virus (SPCSV) which was shown to be involved in downregulation of RNAi. SPCSV RNase III belongs to the Class 1 enzymes, able to degrade siRNA to effectless small RNA. In the present investigation we compared the silencing suppression activity of a putative RNase III Class 1 protein of pike perch iridovirus (PPIV: Iridoviridae, Ranavirus, predominantly infecting fish species) to that of the previously characterized SPCSV RNase III infecting sweetpotato. The RNase III-like gene of PPIV encodes a functional RNase III enzyme of about 40.2 kDa, which has dsRNA-specific endonuclease activity and was able to degrade long dsRNA and short siRNA in vitro. In addition, the capacity of silencing suppression of SPCSV RNase III and PPIV RNase III and their mutated forms was assessed in suppression experiments in Nicotiana benthamiana. Interestingly, expression of wild type PPIV RNase III enzyme in agroinfiltrated leaves of N. benthamiana plants correlated with suppression of RNA silencing. The mutant with comprised activities showed reduced silencing suppression capacity.

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P19 EPIDEMIOLOGY AND MANAGEMENT OF COCONUT LETHAL YELLOWING DISEASE IN CENTRAL REGION OF MOZAMBIQUE João Bila1, Ana Mondjana2, Berit Samils1 and Nils Högberg1 1 Swedish University of Agricultural Sciences, Uppsala, Sweden, 2Universidade Eduardo Mondlane, Maputo, Mozambique

Abstract Coconuts and coconut products form an important part of the economy in Mozambique. However, outbreaks of coconut lethal yellowing disease (CLYD) now threaten the industry and the livelihood of over 1.7 million people in Mozambique. Affected trees cease producing and threaten the productivity of healthy trees. Quarantine measures including roadside checkpoints are in place to restrict movement of coconut planting materials from north to south of Mozambique. Where the disease is endemic, feeling of infected coconut tree is the unique control measure applied. In order to minimize the impact of the disease, this research project is proposed. The overall objective is to improve the livelihood of the coconut producers and the coconut secondary products users by 2015 due to generation of coconut lethal yellowing disease epidemiology new knowledge attempting to implement sustainable disease management approach in the central region of Mozambique. The general objective will be addressed through the following specific objectives: (i) To characterize the phytoplasma species, causal agent of coconut lethal Yellowing disease (CLYD) in central region of Mozambique; (ii) To evaluate the efficiency of the suspected tree removal (feeling) method in the management of CLYD; (iii) To investigate the potential of seed transmission of CLYD; (iv) To investigate the potential insect vectors of the coconut lethal yellowing pathogen in Mozambique; and finally (v) To identify the diversity of the plant secondary hosts of the phytoplasma species causing CLYD in central Mozambique. Provided that phytoplasma are obligate organisms, the laboratory work will rely on molecular technique. The expected outputs of the project are several manuscripts dealing with epidemiology and management of coconut LY disease in central Mozambique. Improvement of livelihood of the coconut stakeholders as result of adoption of sustainable disease management approach is the main outcome to be generated by the project.

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