Poster Category  7:


Fungal and Oomycete Effectors



GEMO: Evolutionary Genomics of Magnaporthe oryzae

Didier Tharreau[6] Kroj T[1] Chiapello H[2] Aguileta G[2] Rodolphe F[2] Gendrault A[2] Amselem J[3] Lebrun MH[4] Fournier E[5]

1INRA, UMR BGPI, TA A54 K, 34398 Montpellier, France, 2INRA, UR MIG, 78352 Jouy-en-Josas, France, 3INRA, UMR BIOGER, 78 850 Thiverval Grignon, France, 4CNRS, UMR BIOGER, 78 850 Thiverval Grignon, France, 5INRA, UMR BGPI, TA 54K, 34398 Montpellier, France, 6CIRAD, TA A 54/K, 34398 Montpellier, France

Developing integrated control methods against pests of cultivated plants can significantly contribute to increasing food production while reducing inputs threatening the environment. The durability of a control method can be improved by a better knowledge of the pathogen’s genetic determinants that are responsible for this adaptation. We were granted by the French National Research Agency for a project that aims at sequencing the genomes of several strains of the phytopathogenic model species Magnaporthe oryzae and at exploiting these complete sequences to characterize the repertoire of genes involved in pathogenicity and host specificity, and study their evolution. We will sequence 7 strains of the species M. oryzae representing different genetic groups pathogenic of different species of Poacees and one strain of the sister species M. grisea. ESTs produced during the infection by two strains pathogenic of rice and wheat on their respective host will also be sequenced. Different available annotation pipelines will permit to list and do comparative analyses of different gene families known or speculated to be involved in pathogenicity. Transcriptomic data of the two strains with different host specificities will be compared to identify key genes in specialization to the host. Genome fluidity will be characterized by synteny analyses and by the identification and localization of repeated elements. The impact of these rearrangements on pathogenicity genes and host specificity genes will be tested. Molecular signatures of positive or purifying selection in coding and regulatory sequences will be searched for by different methods. The whole set of data will be integrated in a database that will be designed to be accessible publicly.




Molecular characterisation of effector proteins from the fish pathogenic oomycete Saprolegia parasitica

Irene de Bruijn, Minor, K.L., Phillips, A.J, Robertson, E.J., Anderson, V.L., Bain, J., Wawra, S., Secombes, C.J, van West, P.

University of Aberdeen

Water molds (oomycetes) are destructive pathogens of aquatic animals and terrestrial plants. Saprolegnia species cause Saprolegniosis, a disease that is characterized by visible white or grey patches of filamentous mycelium on the body or fins of freshwater fish. Saprolegnia parasitica is economically one of the most important fish pathogens, especially on catfish, salmon and trout species, causing millions of dollar losses to the aquaculture business worldwide. Several Saprolegnia species have also been linked to declining wild fish stocks and amphibian populations around the world. Currently, the genome of S. parasitica (isolate CBS223.65) is being sequenced by combined Sanger, 454 and Illumina data. The annotation is supported by paired-end EST sequences. Analysis of the preliminary genome sequence and EST libraries resulted in the identification of putative effector proteins. Electron microscope analysis showed that Saprolegnia interacts with fish cells by forming haustoria-like structures. Detailed expression studies of the genes encoding the putative effectors were performed during the biotrophic and necrotrophic infection stages of S. parasitica. Also localization and uptake studies were performed to show a role of the effector proteins in the interaction of S. parasitica with a rainbow trout cell-line.



Genome sequence of anamorphic basidiomycetous yeast Pseudozyma antarctica

Tomotake Motita, Hideaki Koike, Masayuki Machida, Tokuma Fukuoka, Tomohiro Imura, Dai Kitamoto

National Institute of Advanced Industrial Science and Technology (AIST)

Pseudozyma antarctica (renamed from Candida antarctica) is an anamorphic basidiomycetous yeast, which is closely related to the corn smut fungus Ustilago maydis. P. antarctica is known to produce the two different lipases, lipase A and B, which are useful biocatalysts with a broad range of industrial applications. P. antarctica was also found to produce itaconic acid, which is used in the manufacture of synthetic resins, coating, and other industrial products. Interestingly, P. antarctica is able to produce a large amount of the extracellular glycolipid biosurfactants, mannosylerythritol lipids (MELs) from a variety of biomass such as vegetable oils, glycerol, and some sugars. MELs show not only excellent surface-active properties but also versatile biochemical actions, and thus have been received increasing attentions as new bio-based materials [1]. Previously, in order to apply genetic engineering to the large-scale production of MELs, we carried out the expressed sequence tags (EST) analysis of P. antarctica T-34, and described the genes expressing under the MEL production conditions [2]. Consequently, we believe that the genomic information of the basidiomycetous yeast should allow us to develop the novel processes for producing many practical bio-products such as proteins, organic acids, sugars, and glycolipids. Furthermore, the comparative analysis of the genome sequences between P. antarctica and U. maydis would give the important knowledge for plant infection mechanism. We thus focused our attention on the genome sequence of P. antarctica. Here, we present the draft genome sequence of P. antarctica, the putative genes derived from the previous result of EST analysis. This study was supported by the Industrial Technology Research Grant Program in 06A17501c from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. [1] Morita et al., Biotechnol Appl Biochem, 53, 39-49, 2009. [2] Morita et al., Yeast, 23, 661-671, 2006.



The whole genome sequence of Venturia inaequalis, the causal agent of apple scab, using next generation sequencing technology: implications for identifying candidate effector genes

Joanna Bowen[1] Ross Crowhurst[1] Carl Mesarich[2] Kim Plummer[3] Matthew Templeton[1]

1The New Zealand Institute for Plant & Food Research, Auckland, New Zealand.
2The New Zealand Institute for Plant & Food Research, Auckland, New Zealand and The University of Auckland, Auckland, New Zealand, 3La Trobe University, Melbourne, Australia

The hemi-biotrophic fungus Venturia inaequalis infects members of the Maloideae, causing the economically important apple disease, scab. The genetics of the interaction between Malus and V. inaequalis follow the gene-for-gene model; effectors (pathogen proteins required for infection) are presumably secreted to the plant/pathogen interface early during the infection cycle where a subset can be recognised by plant resistance gene products to induce a hypersensitive response. Previously, expressed sequence tag and proteomic approaches were adopted to identify candidate effector genes but were limited in their success by the extent of sequence coverage. Therefore, sequencing the whole genome of an isolate of V. inaequalis theoretically carrying a full complement of effectors was performed using Illumina technology. A total scaffold length of 36Mb was assembled at an estimated 96% coverage of genes calculated by mapping 131 SSR markers and ESTs. Orthologues of several fungal effector genes were identified including Ecp6, AvrLm6 and Avr-Pita.  Previously, using reverse genetics, we had obtained amino acid sequence information from a candidate AvrRvi4,5 effector that elicited a hypersensitive response on hosts 4 and 5.  This sequence exactly matched part of a predicted novel 154-amino acid protein in the whole genome sequence which has a leader sequence and eight cysteines. Functional analysis of this and other candidate genes is currently underway by silencing/disruption. Further sequencing, including transcriptome analysis and mate-end paired reads, will be carried out to reduce the number of scaffolds in the assembly and to aid annotation.


Functional analysis of Six proteins: effectors of Fusarium oxysporum

Petra Houterman, Fleur Gawehns, Caroline Michielse, Ringo van Wijk, Lisong Ma, Myriam Clavijo Ortiz, Ines Schreiver, Ernst-Jan Eggers, Charlotte van der Does, Ben Cornelissen, Frank Takken, Martijn Rep,

Universiteit van Amsterdam, The Netherlands


The plant xylem-colonizing fungus Fusarium oxysporum f.sp. lycopersici (Fol) secretes small proteins into xylem sap during colonization of its host, tomato. We call these small proteins ‘Six’ proteins for ‘Secreted in xylem’. Through gene knock-out and complementation we established that several Six proteins are required for full virulence. In addition, three Six proteins trigger R gene-dependent immunity and are therefore called Avr1, Avr2 and Avr3.


Some SIX genes are activated specifically upon entry into roots, and expression of all SIX genes investigated is fully dependent on the transcription factor Sge1 (‘Six gene expression 1’). In accordance with this, strains deleted for SGE1 are non-pathogenic and are inhibited in growth inside tomato roots.


Results of transient expression assays in leaves of Nicotiana benthamiana suggest that some Six proteins can suppress disease resistance reactions (hypersensitive response) while others enhance these reactions. Our aim is to uncover the molecular mechanisms underlying these effects.





Chasing effectors in the secretome of Melampsora larici-populina, the causal agent of poplar leaf rust

Stéphane Hacquard[1], Joly D L[2], Lin Y-C[3], Tisserant E[1], Frey P[1], Hamelin R[2], Martin F[1], Duplessis S[1]

1UMR Interactions arbres-Microorganismes, INRA Nancy, Champenoux, France
2Laurentian Forestry Centre, Natural Resources Canada, Québec, Canada
3 VIB, Department of Plant Systems Biology, Ghent, Belgique

The foliar rust caused by Melampsora larici-populina is the main disease affecting poplar plantations in Northern Europe with severe economic losses. In the wake of the Populus genome sequencing, the ~100 Mb genome of M. larici-populina have been sequenced (7X depth) by the Joint Genome Institute (JGI, Department of Energy, USA). The analysis of this genome is a great opportunity to identify loci coding for small-secreted proteins (SSP) produced by the rust fungus to penetrate and exploit its host. The genome sequence of M. larici-populina has revealed a large arsenal of approximately two thousand secreted proteins, half corresponding to SSP (≤ 300 aa). Similarities with effectors previously described in Pucciniales were also uncovered, such as homologs of avirulence factors from Melampsora lini or of the Rust Transferred Protein RTP1 from Uromyces fabae. Transcriptome analyses based on 454-pyrosequencing and NimbleGen systems oligonucleotide arrays allowed to identify transcripts encoding SSP specifically and highly expressed during parasitic growth. Expression profiles of these candidates were monitored by RT-qPCR in M. larici-populina during infection of poplar leaves, showing a preferential expression after haustorium formation. Immunolocalization of selected SSP containing RxLR-like motif using confocal laser scanning microscopy indicated accumulation around haustorial structures, likely in the extra-haustorial matrix. Such approaches led to the identification of candidate rust effectors, for which functional characterisation is ongoing.




Identification and characterization of candidate effector proteins secreted by the crucifer anthracnose fungus Colletotrichum higginsianum

Jochen Kleemann, Richard O'Connell

Max-Planck-Institute for Plant Breeding Research

The hemibiotrophic ascomycete Colletotrichum higginsianum causes anthracnose disease on brassica crops and the model plant Arabidopsis thaliana. Successful plant infection requires the development of a specialized infection structure called an appressorium and the establishment of biotrophic hyphae inside living epidermal cells. We hypothesize that appressoria and biotrophic hyphae secrete effector proteins that permit the fungus to evade or disarm host defence responses and to reprogram host cells. As a first step towards the discovery of secreted effectors in C. higginsianum, we have generated expressed sequence tags (ESTs) from appressoria grown in vitro, isolated biotrophic hyphae and appressoria piercing the epidermal cell wall. Biocomputational prediction tools were used to identify small, soluble secreted proteins showing no homology to known proteins – hallmarks of many microbial effectors. Colletotrichum higginsianum effector candidates (ChECs) that were upregulated at developmental stages relevant to the establishment of biotrophy were selected for functional analysis. This included targeted gene disruption in C. higginsianum, localization in infected plants by over-expression of tagged proteins, and transient in planta expression assays to assess the ability of selected effectors to suppress elicitor-induced cell death. ChEC3 and its paralogue ChEC3a, both displaying weak similarity to C. gloeosporioides CgDN3, were found to suppress necrosis induced by C. higginsianum Nep1-like protein 1 (ChNLP1). Effector ChEC4, containing a predicted nuclear localization signal, was found to target the plant nucleus when expressed in planta as a GFP fusion protein suggesting that this effector has the potential to interfere with host gene expression.

biotrophy, Colletotrichum higginsianum, Effectors, expressed sequence tags ,




Genome mining and functional genomics of small secreted proteins (SSPs) in Cladosporium fulvum, Mycosphaerella graminicola and M. fijiensis

Rahim Mehrabi[1] Amir Mirzadi[1] Gert H.J. Kema[1] Pierre P.J.G.M. de Wit[2]

1Plant Research International BV, Wageningen University & Research Centre, Droevendaalsesteeg 1, 6708PB, Wageningen, The Netherlands
2Laboratory of Phytopathology, Wageningen University & Research Centre, Droevendaalsesteeg 1, 6708PB, Wageningen, The Netherlands

Fungal pathogens secrete effector proteins to target and manipulate host plants for successful infection. They primarily function as virulence factors but during co-evolution plants have evolved resistance proteins to recognize them resulting in effector-triggered immunity (ETI). Successful pathogens, in turn, evolved additional effectors to evade recognition or to suppress ETI resulting in host susceptibility. In this study we mined the genomes of Cladosporium fulvum, Mycosphaerella graminicola and M. fijiensis and identified 289, 266 and 180 SSPs, respectively, that represent a common feature of effector proteins (<300 aa residues and containing ≥4 cysteine residues). We used a combined proteomics and expression profile approach and selected over 100 SSPs for further functional analysis.  The SSPs are cloned into an Agrobacterium tumefaciens overexpression vector with or without the PR-1A signal peptide sequence for transient expression in planta.  We are testing the effect(s) of SSPs in suppression of HR or necrosis induced by Inf1, CfHNNI1, BAX or Cfs/Avr gene pairs in tobacco plants. The role of some promising candidates in virulence will be assayed by gene knock-out studies.  In addition, we will produce M. graminicola SSPs in an Escherichia coli heterologous expression system and purify them using FLAG affinity purification. Subsequently, M. graminicola SSPs will be infiltrated in a set of differential cultivars to identify SSPs that are able to cause HR or necrosis. Systemic overexpression of M. graminicola SSPs will be performed using the BSMV-mediated expression system in wheat differentials. Preliminary data and comparison of SSPs from the different fungal pathogens will be discussed. 

Rahim Mehrabi is financially supported by an ERA-PG grant (ERA-PG 31855.004) and  Pierre JGM de Wit by the Royal Netherlands Academy of Arts and Sciences.





Characterisation of two necrosis and ethylene inducing protein-like (Nlp) genes from Sclerotinia sclerotiorum

Kim Plummer[1] Adrian Dinsdale[1] Floor Van Den Elsen[2] Rebecca Barnett[1]

1La Trobe University, 2Wageningen UR Plant Breeding, 6700 AJ Wageningen, The Netherlands

The necrotrophic phytopathogen, Sclerotinia sclerotiorum, induces plant cell death in order to colonise host plants and release nutrients. This pathogen is known to secrete various compounds, including oxalic acid and lytic enzymes during infection.  Infection is often facilitated in other necrotrophs (such as Fusarium oxysporum and Botrytis cinerea) by the secretion of small, phytotoxic effector molecules (including necrosis and ethylene inducing peptides, NEPs). We have cloned two genes from S. sclerotiorum with significant similarity to NEPs called NEP-like1 (Nlp1) and Nlp2.  Both NLPs appear to induce necrosis when transiently expressed in Nicotiana tobaccum and N. benthamiana. Multiple 35S fusion constructs, designed to investigate the effect of in planta NLP protein expression (with or without signal peptides) have also been completed and transformations are currently underway.  Both genes have also been expressed in Pichia pastoris resulting in purified NLP1 and NLP2 that will be used for further transient assays to assess the specific response induced in host plants when presented with one or both proteins. Multiple Nlp1RNAi mutant lines have been generated which display inhibited growth rates both in vitro and in planta; further characterisation of these will be presented.  Expression localisation studies are also underway using NLP promoter:GFP fusion constructs in transformed S. sclerotiorum.


Identification of a Sporisorium reilianum effector involved in host specificity

Theresa Wollenberg[1] Katja Zuther[1] Stephan Poppe[2] Regine Kahmann[2] Jan Schirawski[1]

1Max-Planck-Institute for Terrestrial Microbiology, 35043 Marburg, Germany; Georg-August-University Göttingen, Albrecht-von-Haller Institute for Plant Sciences, 37073 Göttingen
2Max-Planck-Institute for Terrestrial Microbiology, 35043 Marburg, Germany

Sporisorium reilianum and Ustilago maydis are smut fungi with a narrow host range. U. maydis and the S. reilianum variety SRM produce smut symptoms only on maize, while the S. reilianum sorghum variety SRS produces spores on sorghum. Microscopic analysis showed that after host plant penetration S. reilianum reaches the inflorescence via growth along the vascular bundles. SRS hyphae can also ramify in maize plants and reach the inflorescence but do not differentiate into spores. We wanted to identify genes that support virulence on maize but hamper virulence on sorghum. Using genome comparison and heterologous PCR/Southern analysis we identified three putative candidate genes that are present in the maize pathogens U. maydis and SRM but absent in the sorghum pathogen SRS. One of these genes, c1, encodes a secreted protein that is highly conserved between U. maydis and SRM. Deletion of c1 in U. maydis drastically reduced virulence. Expression of c1 of SRM in SRS led to strains that more efficiently reached the inflorescence as deduced by an increased incidence of phyllody – a typical symptom associated with S. reilianum infection. This shows that the identified effector functions as a virulence factor for maize.


Expression of c1 in SRS led to the appearance of colored spots on veins of infected sorghum plants indicating a host defense response. In addition, virulence on sorghum was reduced, suggesting that c1 functions as a virulence-attenuating factor for sorghum. Thus, the identified effector contributes to host specificity of S. reilianum.





Hunting for effectors-elicitors in the fungal wheat pathogen Mycosphaerella graminicola with a quantitative proteomic approach using comparative (label-free) LC-MSE  
Sarrah Ben M’Barek1, Cordewener, JHG1,2,3, Van der Lee, TAJ1, America, AHP1,2,3, Kema, GHJ1
1 Plant Research International, Wageningen UR, PO box 69, 6700AB Wageningen
2 Center for BioSystems and Genomics, 6700AA, Wageningen, the Netherlands
3 Netherlands Proteomics Centre, Utrecht, The Netherlands

Septoria tritici blotch caused by the haploid ascomycete Mycosphaerella graminicola (Fuckel) J. Schröt. in Cohn, is the most important wheat disease in Europe. Despite the recent identification of 15 resistance genes and their potential application in breeding, disease control is currently achieved mainly by fungicides. The genome of M. graminicola has been sequenced and finished by the US. Department of Energy- Joint Genome Institute and together with the high quality of the genome annotation, the high-density genetic linkage maps, including 11 quantitative trait loci involved in species specificity as well as in cultivar-specificity (Ware et al. 2006; Wittenberg et al. 2007) this provides an excellent foundation for proteomic studies that focus on the pathogen side of the interaction. The lifestyle of M. graminicola is significantly different from other cereal pathogens. It is a hemibiotroph with an initial symptomless biotrophic and intercellular phase that is followed by a necrotrophic phase resulting in disease symptoms starting from approximately 14 days after infection. The switch from biotrophy to necrotrophy is poorly understood and we therefore have started experiments using established protocols for apoplast analysis from the Cladosporium-tomato pathosystem. Extracellular liquid (apoplast) was collected from (in)compatible interactions at several time points after inoculation. Proteins were extracted and digested with trypsin. The complex peptide digests were separated and detected with nano-UPLC-QTOF operating in an alternating mode of low and high collision energy. With this approach peptide abundances can be quantitatively compared between multiple complex protein samples. In the initial experiment 18 LCMS traces (6 samples in triplicate) were compared providing highly detailed quantification and identification. Using the identification algorithm 3150 peaks were identified, resulting in 1926 unique peptide sequences that were identified and quantified. We identified several proteins of M. graminicola that appear to be secreted in specific stages of the infection to the apoplast and currently study these candidate effectors of the M.graminicola-wheat interaction.





Trehalose synthesis in Aspergillus niger and other fungi

Petter Melin, Åsa Svanström

Swedish University of Agricultural Sciences


The disaccharide trehalose has several important roles in fungi, e.g. in stress protection, spore germination and virulence. It also serves as a major component in asexual and sexual spores. In order to reveal the exact function of this extraordinary sugar, it is essential to identify and reveal the functions of the enzymes involved and how these are regulated. In filamentous fungi some components have been identified, mostly in Aspergillus nidulans, but no overall characterization has been performed. In Saccharomyces cerevisiae the four enzymes behind the pathway from glucose to trehalose are characterized. Using bioinformatics we found that in other yeasts and in filamentous fungi the protein composition differs substantially. In some sequenced Aspergilli, including A. niger, A. flavus and A. fumigatus six homologues proteins were identified. We call the enzymes with proposed trehalose phosphate synthase activities TpsA, TpsB and TpsC. Three proteins with believed phosphatase activities are called TppA, TppB and TppC. We have started a comprehensive characterization of all enzymes using A. niger as target organism. In most deletion mutants, the content of trehalose was initially reduced but after maturation there were no significant differences compared to wildtype. Two notable exceptions: In the tppA deletion mutant only abnormal conidiophores were formed and in the very few conidia we could detect only traces of trehalose. The other exception, the tppB mutant, had wildtype-like conidiephores but with a severely reduced conidial trehalose content. This mutant is a usable tool to reveal the role of trehalose in spore germination and survival.





The transcriptome of an intracellular hemibiotroph, Colletotrichum higginsianum

Richard O'Connell, Hiroyuki Takahara, Jochen Kleemann

Max Planck Institute for Plant Breeding Research

Colletotrichum higginsianum causes anthracnose disease on Arabidopsis thaliana, providing a model pathosystem in which pathogen and host genomes are available and both partners can be genetically manipulated. After initial penetration by appressoria, the fungus grows biotrophically in living epidermal cells, producing bulbous hyphae that invaginate the host plasma membrane, before entering a destructive necrotrophic phase. To survey fungal gene expression during biotrophy, we made a stage-specific cDNA library from hyphae isolated from infected leaves by fluorescence-activated cell sorting. The high purity of the isolated hyphae eliminates contamination by transcripts from host cells or other fungal cell types. EST sequencing showed that genes related to redox homeostasis, biosynthesis of amino acids and vitamins and the uptake of amino acids, mono- and disaccharides were well-represented. To search for fungal effectors, we used computational prediction tools to identify genes encoding small, soluble secreted proteins. Expression profiling showed that many candidate effector genes are plant-induced and highly stage-specific. One such gene, CIH1, encodes a protein with two LysM chitin-binding domains that accumulates at the biotrophic interface and may function in PAMP concealment. Targeted gene disruption suggested that CIH1 is required for the establishment of intracellular biotrophic hyphae. In Nicotiana benthamiana transient expression assays, one candidate effector induced plant cell death, while six others suppressed necrosis induced by a C. higginsianum Nep1-like protein 1. Overall, our results suggest that Colletotrichum biotrophic hyphae are organs for both nutrient acquisition and effector delivery.





Secreted oomycete proteins in Phytophthora rot of garden pea

Doris Roth[2] Fredrik Heyman[1] Morten Nedergaard Grell[2] Dan Funck Jensen[1] Lene Lange[2]

1Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences
2Section for Sustainable Biotechnology, Copenhagen Institute of Technology/Aalborg University

Host-pathogen interactions rely on secreted proteins. Studying the secretome is therefore a suitable way to identify genes implicated in pathogenicity. Here, we present a project to investigate the interaction between pea and a root-rot pathogen, a mostly uncharacterized Phytophthora sp., by identifying proteins secreted by pea and oomycete during infection. To this end, Phytophthora sp.-infected pea roots are used to produce dual organism cDNA libraries. These libraries are screened for cDNA sequences encoding relevant proteins that are then further characterized. Of interest are both enzymes to breakdown tissue into nutrients and other proteins that function directly in virulence. Through the secretome studies we will deepen the understanding of the novel Phytophthora species and its function as causal agent of an economically important pea disease.





Functional analysis of homologues of the Cladosporium fulvum Avr4 and Ecp2 effectors present in other (pathogenic) fungal species

Ioannis Stergiopoulos[1] Henriek Beenen[1] Harrold van den Burg[1] Yiannis A.I. Kourmpetis[2] Bilal Okmen[1] Pierre J.G.M. De Wit[1]

1Laboratory of Phytopathology, Wageningen University & Research Centre, Droevendaalsesteeg 1, 6708PB, Wageningen, The Netherlands
2Department of Biometris, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands

Cladosporium fulvum is a non-obligate biotrophic fungus of the Dothideomycetes class that causes leaf mould of tomato. During infection, C. fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce effector-triggered immunity in their presence. Recently, homologues of the C. fulvum Avr4 and Ecp2 effectors were identified in species of Dothideomycetes, including Mycosphaerella fijiensis the causal agent of the black Sigatoka disease of banana. We have demonstrated that the M. fijiensis Avr4 is a functional orthologue of the C. fulvum Avr4 that binds to chitin and triggers a Cf-4-mediated hypersensitive response (HR) in tomato, suggesting that a common recognition site in the two effectors is recognized by the Cf-4. Using a targeted mutational approach, we are examining whether the chitin-binding domain present in these two effectors represents this recognition site. Three homologues of the C. fulvum Ecp2 are found in M. fijiensis, two of which induce different levels of necrosis or HR in tomato lines that lack or contain a cognate Cf-Ecp2 protein. Therefore, Ecp2 is suggested to promote virulence by interacting with a putative host target, causing host cell necrosis. Using a yeast-two-hybrid assay we will try to isolate Ecp2-interactors from tomato to further unravel the role of this effector in virulence. Finally, we are expanding our searches for Avr4 and Ecp2 homologues in fungal species outside the class of Dothideomycetes. Preliminary data suggest that Ecp2 is widely distributed among fungal species but has significantly diverged after speciation of these fungi.






Dissecting the role of Cladosporium fulvum (secreted) proteases and protease inhibitors

Mansoor Karimi Jashni, Rahim Mehrabi, Harrold A. van den Burg, Pierre J.G.M. de Wit,

Laboratory of Phytopathology, Wageningen University & Research Centre, Droevendaalsesteeg 1, 6708PB, Wageningen, The Netherlands


In order to facilitate infection, fungal pathogens produce various types of secreted proteases likely to target and perturb important plant proteins that are involved in controlling basal defense. In addition, they secrete several protease inhibitors such as Avr9 of Cladosporium fulvum that, based on its structure, is predicted to be a carboxy peptidase inhibitor. Protease inhibitors are potentially able to deactivate or detoxify host target proteases and, therefore, might play an important role during infection. In this study we are investigating the role of C. fulvum protease and protease inhibitors in disease establishment by using both functional genomics and biochemical approaches. We mined the genome of  C. fulvum and found numerous proteases and protease inhibitors of which many are secreted. Expression analyses of these genes were performed using RNA extracted from fungal mycelium grown in vitro on liquid media under different conditions as well as from inoculated susceptible tomato plants.  Interestingly, many of these genes are highly expressed only in vitro and/or in planta and based on their expression profiles we selected a number of candidates for further functional analyses.  We will generate knock-out mutants of the selected proteases and protease inhibitors to identify their role in virulence. In addition, biochemical approaches will be used to pull-down the host target proteins of some presumably important candidate proteins such as for example the Avr9 protein.





Identification and functional characterization of Cladosporium fulvum effectors by genomics, transcriptomics and proteomics approaches

Bilal Okmen[1] Ioannis Stergiopoulos[1] Mattias De Hollander[2] Harrold van den Burg[1] Henriek Beenen[1] Pierre J.G.M. De Wit[1]

1Laboratory of Phytopathology, Wageningen University & Research Centre, Droevendaalsesteeg 1, 6708PB, Wageningen, The Netherlands
2Applied Bioinformatics, Plant Research International, PO Box 16, 6700AA Wageningen, The Netherlands

Cladosporium fulvum is a biotrophic fungal pathogen that causes leaf mold of tomato. During infection C. fulvum secretes a number of small proteins into the apoplast of tomato leaves, which are collectively called effectors. So far, ten effector proteins have been characterized that in general show no or limited sequence similarity to other proteins present in public databases. In this study we try to identify and functionally characterize additional C. fulvum effector proteins that are involved in fungal pathogenesis. Recently, the genome of C. fulvum has been sequenced using the 454 technology. This genome sequence enables the identification of all secreted proteins from the fungus, collectively called the secretome. However, for accurate mining and annotation of the effector secretome, gene calling programs need to be first optimized by analyzing high quality expressed sequence tags (ESTs). Therefore, several cDNA libraries of C. fulvum grown under various in vitro and in planta conditions were constructed and are sequenced to support genome annotation. Initial automated annotation of the genome revealed that the fungus contains approximately 13.000 genes, of which approximately 1200 encode putatively secreted proteins. Bioinformatic analyses identified a subset of 300 putative effectors within the predicted secretome, while additional proteomics analysis from apoplastic fluids of tomato leaves infected by C. fulvum, revealed 30 proteins that are specifically produced in the compatible interaction. At this moment we are performing functional profiling of these novel effector proteins by examining their ability to inhibit PAMP triggered Immunity and/or effector-triggered immunity in custom made assays.



Identification and analysis of secreted proteins from the maize pathogen Colletotrichum graminicola

Jorrit-Jan Krijger, Fabian Weihmann, Christian Kröling, Holger B. Deising, Stefan Wirsel

Martin-Luther-Universität Halle-Wittenberg

The filamentous Ascomycete Colletotrichum graminicola is the causal agent of stem rot and leaf anthracnose on Zea mays. After germination and penetration of epidermal cells, this hemibiotrophic fungus enters a short biotrophic phase that is followed by a destructive necrotrophic phase resulting in the production of conidia. Secreted fungal proteins are believed to play important roles in the progress of both phases of pathogenesis. The Yeast Secretion Signal Trap (YSST) was used to identify cDNAs encoding peptides containing signal sequences, starting from mRNA from in vitro-grown mycelium, induced with a corn leaf-extract. Of the 94 obtained sequences, 45 showed significant similarities to genes with a reported function, 24 were similar to genes annotated in fungal genome projects and 27 showed no similarity to database entries. Macroarray hybridisation showed that most of these genes are expressed in planta. Transcript abundance of most genes peaks at specific periods during pathogenesis, while some are expressed constantly. A minor set exhibits two peaks and a minimum during the biotrophy-necrotrophy transition phase. Expression patterns of several genes from each set were confirmed by qRT-PCR.

To test possible roles of secreted proteins as pathogenicity or virulence factors, knock-out strains were generated for 18 genes identified in the screen, encoding proteins of various classes: enzymes, small cystein-rich proteins and proteins of unknown function. Results from infection assays will be presented.





Crystal structure of the avirulence gene AvrLm4-7 Of Leptosphaeria maculans illuminates its evolutionary and functional characteristics

Isabelle Fudal[1] Françoise Blaise[1] Karine Blondeau[2] Marc Graille[2] Audrey Labarde[2] Anthony Doizy[2] Brett M. Tyler[3] Shiv D. Kale[3] Guillaume Daverdin[1] Marie-Hélène Balesdent[1] Herman van Tilbeurgh[2] Thierry Rouxel[1]

1UMR Bioger-CPP, INRA Grignon, France, 2IBBMC-CNRS, Université Paris-Sud, Orsay, France
3Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, USA

Leptosphaeria maculans, a phytopathogenic ascomycete causing stem canker of oilseed rape, develops “gene-for-gene” interactions with its host plant where fungal avirulence (AvrLm) genes are the counterpart of plant resistance (Rlm) genes. AvrLm4-7 encodes a 143 amino-acid cysteine-rich protein, secreted outside of the fungus cells and strongly induced during the early stages of plant infection. AvrLm4-7 can bind to phosphoinositides and is translocated within plant cells. AvrLm4-7 crystal structure was determined following heterologous production in Pichia pastoris. The protein shows the presence of 4 disulfide bridges, and is strongly positively charged, suggesting interaction with minus charged molecules such as DNA or phospholipids. AvrLm4-7 confers a dual specificity of recognition by Rlm7 or Rlm4 resistance genes and occurs as three alleles only: the double avirulent (A4A7), the avirulent towards Rlm7 only (a4A7), or the double virulent (a4a7). Sequencing of diverse alleles coupled with targeted point mutagenesis strongly suggested that one single base mutation, leading to the change of a glycine residue to an arginine, was responsible for the A4A7 to a4A7 phenotype change. This amino acid being located on an external loop of the protein, its change is unlikely to alter the 3-D structure of the protein, but rather must correspond to a specific recognition target for the plant cell. In contrast, multiple mechanisms are responsible for the complete loss of avirulence (a4a7 phenotype), mostly drastic events leading to inactivation or complete deletion of the gene. The few single point mutations found targeted amino acids essential for the 3-D structure of the protein.


Functional characterization of LaeA

Graeme S. Garvey, Jonathan M. Palmer, Jin Woo Bok, Alex J. La Reau, Nancy P. Keller

University of Wisconsin-Madison

Here we present the initial findings of a biochemical and genetic investigation into the mechanism of LaeA, a putative methyltransferase that functions as a global regulator of secondary metabolism in Aspergillus nidulans. LaeA has been found to be part of a large nuclear velvet complex that is required for secondary metabolite production as well as light regulated morphological development. Preliminary data suggests LaeA may control secondary metabolite gene clusters through chromatin remodeling. However, there is no direct evidence linking the velvet complex to chromatin remodeling. We have initiated a study to functionally characterize LaeA. Several LaeA orthologs were recombinantly expressed in E. coli and assayed for solubility. The full length LaeA protein from A. nidulans is only soluble as a MBP fusion protein, which has proved to be uninformative for in vitro activity assays. A partial proteolysis study was performed to identify soluble domains that could be amenable to in vitro analysis. Soluble truncation mutants were identified and have proved useful for in vitro methyltransferase activity assays. Validation of the truncated LaeA proteins was carried out through successful in vivo complementation of a ∆laeA mutant. These truncation mutants are functionally equivalent to the full-length protein by restoring sterigmatocystin (ST) biosynthesis to wild type levels. Using the truncated LaeA protein, we have confirmed binding of S-adenosyl-L-methionine (methyl group donor) and have identified a methyltransferase activity. Each candidate protein substrate’s site of methylation is being mapped by trypsin digestion coupled with LC/MS. The in vivo role of LaeA methylation will be evaluated with point mutants for effects on ST biosynthesis. Our findings confirm LaeA has methyltransferase activity and provide the first functional insights into the mechanism of LaeA regulation of secondary metabolism.



Cloning and characterization of SnTox1, a novel virulence effector gene important in the wheat-Stagonospora nodorum interaction

Tim Friesen[4] Zhaohui Liu[1] Richard P. Oliver[2] Peter S. Solomon[3] Shunwen Lu[4] Justin D. Faris[4]

1Department of Plant Pathology, North Dakota State University, Fargo, North Dakota 58105, USA.
2Australian Centre for Necrotrophic Fungal Pathogens, Western Australian State Agricultural Biotechnology Centre, Division of Health Science, Murdoch University, Western Australia 6150, Australia.
3Plant Cell Biology, Research School of Biological Sciences, The Australian National University, Canberra ACT 0200, Australia, 4US Department of Agriculture-Agricultural Research Service (USDA-ARS), Cereal Crops Research Unit, Red River
Valley Agricultural Research Center, Northern Crop Science Lab, Fargo, North Dakota 58105, USA.

Stagonospora nodorum blotch caused by S. nodorum (teleomorph Phaeosphaeria nodorum) is a destructive disease of wheat that causes significant yield and quality losses worldwide.  Although this disease is a major concern for breeders, it has been difficult to characterize the interaction due to its quantitative nature.  We have shown that the quantitative nature of the S. nodorum-wheat pathosystem is due at least in part to a complex of proteinaceous host-selective toxins (virulence effectors) that interact either directly or indirectly with dominant host sensitivity/susceptibility gene products in an inverse gene-for-gene manner. SnToxA, which was originally identified in Pyrenophora tritici-repentis as well as SnTox1, SnTox2, SnTox3, and SnTox4 have each been shown to be highly important in disease development in the presence of the corresponding dominant wheat sensitivity genes, Tsn1, Snn1, Snn2, Snn3 and Snn4, respectively. The SnTox1-Snn1 interaction was the first to be identified and this interaction was shown to account for as much as 58% of the disease development. Recently, we have cloned the gene responsible for the production of the SnTox1 protein.  The mature SnTox1 is highly cysteine rich and predicted to be approximately 10 kDa. As expected, the immature protein contains a 20 amino acid predicted signal sequence. The cloning of SnTox1 adds another piece of information to this already fascinating system of virulence effectors produced by S. nodorum.



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