Thursday April 1

Parallel session 7: Fungal and Oomycete Effectors

PS7.1

Fungal secondary metabolites as effectors of pathogenicity: role in the complex interplay between rice and Magnaporthe grisea

Jerome Collemare, Rahadati Abdou, Marie-Jose Gagey, Zhongshu Song¹, Walid Bakeer¹, Russell Cox¹, Elsa Ballini², Didier Tharreau², Marc-Henri Lebrun³

1School of Chemistry, Bldg 77, University of Bristol, Bristol BS8 1TS, UK

2UMR BGPI, CIRAD-INRA-SupAgro, Baillarguet TA 41/K, 34398 Montpellier cedex 5, France

³UMR5240 CNRS-UCB-INSA-BCS, CRLD Bayer Cropscience, 69263 Lyon Cedex 09, France

mhlebrun@versailles.inra.fr

Functional analyses of fungal genomes are expanding our view of the metabolic pathways involved in the production of secondary metabolites. These genomes contains a significant number of genes encoding key biosynthetic enzymes such as polyketides synthases (PKS), non-ribosomal peptide synthases (NRPS) and their hybrids (PKS-NRPS), as well as terpene synthases (TS). Magnaporthe grisea has the highest number of such key enzymes (22 PKS, 8 NRPS, 10 PKS-NRPS, and 5 TS) among fungal plant pathogens, suggesting that this fungal species produce a large number of secondary metabolites. In particular, it has 10 hybrid PKS-NRPS that likely produce polyketides containing a single an amino-acid. Three of them (ACE1, SYN2 and SYN8) have the same expression pattern that is specific of early stages of infection (appressorium-mediated penetration), suggesting that the corresponding metabolites are delivered to the first infected cells. M. grisea mutants deleted for ACE1 or SYN2 by targeted gene replacement are as pathogenic as wild type Guy11 isolate on susceptible rice cultivars. Such a negative result could result from a functional redundancy between these pathways. However, ACE1 null mutants become specifically pathogenic on resistant rice cultivars carrying the Pi33 resistance gene compared to wild type Guy11 isolate that is unable to infect such rice cultivars. Introduction of a Guy11 wild type ACE1 allele in Pi33 virulent M. grisea isolates restore their avirulence on Pi33 resistant rice cultivars, showing that ACE1 behaves as a classical avirulence gene (AVR). ACE1 differs from other fungal AVR genes (proteins secreted into host tissues during infection) as it likely controls the production of a secondary metabolite specifically recognized by resistant rice cultivars. Arguments toward this hypothesis involve the fact that the protein Ace1 is only detected in the cytoplasm of appressoria and is not translocated into infectious hyphae inside epidermal cells. Furthermore, Ace1-ks0, an ACE1 allele obtained by site-directed mutagenesis of a single amino acid essential for the enzymatic activity of Ace1, is unable to confer avirulence. According to this hypothesis, resistant rice plants carrying Pi33 are able to recognize its fungal pathogen M. grisea through the perception of one fungal secondary metabolite produced during infection. The map based cloning of the Pi33 rice gene was initiated and this gene maps at a locus rich in classical NBS-LRR resistance genes. Further work is ongoing to identify which gene is Pi33. In order to characterize the secondary metabolite produced by ACE1, this gene was expressed in a heterologous fungal host such as Aspergillus oryzae under the control of an inducible promoter. The removal of the three introns of ACE1 allowed the expression of the enzyme in A. oryzae. Characterization of the novel metabolite produced by Ace1 is in progress.

PS7.2

Effectors of Fusarium oxysporum

Martijn Rep, Petra Houterman, Fleur Gawehns, Lisong Ma, Mara de Sain, Myriam Clavijo Ortiz, Charlotte van der Does, Frank Takken, Ben Cornelissen

University of Amsterdam

m.rep@uva.nl

The tomato xylem-colonizing fungus Fusarium oxysporum f.sp. lycopersici (Fol) secretes effectors into xylem sap of its host. Three of the eleven small secreted proteins that we identified trigger effector-mediated immunity: Avr1, Avr2 and Avr3 are recognized by the resistance proteins I, I-2 and  I-3, respectively. Interestingly, Avr1 suppresses I-2 and I-3-mediated resistance.

Several Fol effectors were shown through gene knock-out to contribute to virulence towards susceptible plants. We aim to uncover the molecular mechanisms through which effectors of Fol trigger susceptibility (suppression of resistance) and immunity (activation of R proteins). We also focus on clarification of genetic processes underlying evolution of host-specific pathogenicity in the Fo species complex.

We found that effector genes in Fol reside on ‘pathogenicity chromosomes’. These chromosomes can be transferred between clonal lines, conferring host-specific pathogenicity to the recipient. Transfer of pathogenicity chromosomes explains the emergence of new pathogenic clonal lines of Fo. This process may also contribute to the evolution of compatibility with novel hosts through recombination between mobile chromosomes resulting in new effector repertoires.

PS7.3

The effectors of Ustilago maydis and related smut fungi

Regine Kahmann

Max Planck Institute for Terrestrial Microbiology, Dept. Organismic Interactions, D-35043 Marburg, Germany

kahmann@mpi-marburg.mpg.de

 The basidiomycete fungus Ustilago maydis is a biotrophic maize pathogen that does not use aggressive virulence strategies and needs the living plant tissue for completion of its life cycle. The U. maydis genome codes for a large set of novel secreted effector proteins. Many of the respective genes are clustered in the genome and are upregulated during pathogenic development. About half of these gene clusters have crucial roles during discrete stages of biotrophic growth. We have now determined which of the clustered effector genes are responsible for the virulence phenotype. We also show that most effectors also exist in related smut fungi, but are poorly conserved, suggesting their involvement in the arms race with the host. U. maydis is eliciting distinct defense responses when individual effector clusters/genes are deleted. Maize gene expression profiling and the identification of interacting proteins allowed us to classify the response to individual mutants and to obtain leads to where the fungal effectors might interfere. Using localization, binding and uptake studies we provide evidence that some effectors function in the apoplast while others are likely to have a cytoplasmic function.

PS7.4

Unraveling the mechanism of RxLR mediated translocation of Oomycete effector proteins

Stephan Wawra^[1] Grouffaud, S^[1] Bain, J.^[1] Whisson, S.C.^[2] Matena, A.^[3] Porter, A.^[1] Bayer, P.^[3] Birch, P.^[2] Secombes, C. J.^[1] van West, P.^[1]

¹University of Aberdeen, ²SCRI Dundee, ³ZMB, Universität Duisburg-Essen, Germany

s.wawra@abdn.ac.uk

Several Prokaryotic and Eukaryotic microbial pathogens have evolved intriguing mechanisms to translocate proteins into their host cells. The translocated proteins are called effectors as they can modulate molecular processes in their hosts in order to establish an infection and/or suppress their immune response. For example, certain types of bacteria possess a needle like injection system, the type III secretion system (T3SS), which allows a direct translocation of effector proteins into the cells under attack. Whilst the bacterial translocation machineries are well described, little is known about how effectors from Eukaryotic pathogens are delivered into their host cells. The early stage of infection caused by the eukaryotic oomycete pathogen Phytophthora infestans involves a biotrophic phase. In this early interaction stage the secretion of oomycete RxLR effectors takes place via haustoria, which are structures formed by the pathogen that are in intimate contact with the extra haustorial membrane produced by the plant. The mechanism by which oomycetes direct their RxLR effectors into host cells is as yet unknown and is the main focus of our research. It has been postulated that endocytosis processes or protein transporters are responsible. Here we present our latest results, which give insight into the mechanism of the oomycete RxLR-EER protein translocation system.

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