1. © 2006 - Cédric Pollet

2. Understanding the Interactions between Biotrophic Fungi & Trees F Martin et al. Tree-Microbe Interactions Joint Unit, INRA/UHP-Nancy US DOE JGI & SHGC Production Sequencing Staffs Genoscope Production Sequencing Staffs Populus Genome Annotation Consortium Laccaria Genome Consortium Tuber Genome Consortium Glomus Genome Consortium Paxillus Genome Consortium Understanding the Interactions between Biotrophic Fungi & Trees F Martin et al. Tree-Microbe Interactions Joint Unit, INRA/UHP-Nancy US DOE JGI & SHGC Production Sequencing Staffs Genoscope Production Sequencing Staffs Populus Genome Annotation Consortium Laccaria Genome Consortium Tuber Genome Consortium Glomus Genome Consortium Paxillus Genome Consortium INRA - AIP Séquençage

3. Genome Sequencing of Micro-organisms Associated to Poplar - the ectomycorrhizal Laccaria bicolor (65 Mb) (released) - the ectomycorrhizal Paxillus involutus (18 Mb) (sequencing in 2008) - the endomycorrhizal Glomus intraradices (18 Mb) (sequencing on-going) - the rust fungus Melampsora larici-populina (120 Mb) (annotation on-going) - the ectomycorrhizal Tuber melanosporum (120 Mb) (annotation on-going) + the mycorrhiza helper Pseudomonas fluorescens BBc6R8 (7 Mb) (annotation on-going) + the weathering soil bacterium Burkholderia (sequencing in 2008) Sequencing of the Populus Community Genome F Martin, P Lammers, A Tunlid & G Tuskan Glomus Melampsora Laccaria Populus Tuber Pseudomonas fluorescens © F Martin - INRA

5. Détection dun ensemble de SNP répartis sur lensemble du génome de Populus nigra V. Jorge, I. Paolucci, A. Bresson, I. Bourgait, S. Schlub, P. Faivre-Rampant AGPF, URGV Objectif : disposer dune large collection de SNP sur 2000 gènes pour des études dassociation et de cartographie, via le génotypage moyen et haut débit. Collaboration avec le Centre National de Génotypage (CNG) + D. Brunel et al. (EPGV) et mutualisation de moyen ave des projets analogues sur vigne et tomate. Stratégie : Choix de 6000 gènes (candidats positionnels et expressionnels / tous caractères, + position aléatoire). Panel de 24 P. nigra (représentatifs de la diversité de lespèce en France). Automatisation de la conception des amorces (Vincent Thareau – IBP Orsay) PCR et séquençage direct sur le produit PCR (amorce universelle - queue SP6 ou T7). Photo: O. Bertel, INRA Orléans

6. saprotrophy symbiosis pathogenesis Plant pathogens Saprophytes Plant Symbionts Animal pathogens Modified from MH Lebrun

7. © INRA Agaricomycotina, Agaricales, Hydnangiaceae, Laccaria bicolor (Maire) P.D. Orton (bicoloured deceiver)

8. Metabolic Zonation/ Physiological compartmentation in a series of hyphal networks Tunlid et al. (2005) New Phytologist, in press Ectomycorrhizal root tips: Symbiosis gene sets Hyphal webs on organic matter: Saprotrophism gene sets Interhyphal Signalling? © Tunlid et al. ECM in decaying leaf materials

9. © INRA Basic features of the Laccaria genome...

10. The Laccaria gene space 37% of the genome is coding sequence 19,128putative proteins (on November 2007) 14,464 with homologs (e-value 0,001) (~ 25% unique to L. bicolor) -------------------- 6,515(~ 34%) covered by Laccaria ESTs 16,741(~ 75%) supported by NimbleGen oligoarrays A large proportion (> 25 %) of Laccaria genes have no homolog in Phanarochaete chrysosporium, Coprinopsis cinerea and other fungi. This group of unique, lineage-specific, genes may include potential loci implicated in symbiotic interactions. 65 Mbp, ~10 chromosomes

11. Expansion of protein families … © INRA

12. 7352 TRIBE families were identified within the sequenced basidiomycetes. The percentage of proteins found in TRIBE families was related to genome size and was largest in L. bicolor. This was mainly due to the expansion of protein family size, but also to a larger number of protein families in L. bicolor as compared to the other basidiomycetes Rajashekar, Ahren, Lin, Duchaussoy et al. Expansion of protein family size & number of protein families

13. The Laccaria secretome: a way to manipulate the host? © INRA

14. Secretome of Laccaria 2,930 proteins with N-terminal secretion signal (14%) 1,625 secreted proteins without TMD domains (49 <AA< 300) 440 unique to L. bicolor 278 with > 6 Cys residues + 48 showed an homology with: – haustoria-expressed secreted proteins (HESP) from the rust fungi Uromyces and Melampsora – pathogenesis-related CFEM-containing proteins from Magnaporthe grisea. >> Effectors interacting with the host plant? J Gibon, F Duchaussoy, F Martin Bioinformatics pipeline identified:

15. NimbleGen Whole Genome Expression Oligoarray -22,294 predicted Laccaria gene models used -178,352 60-mer (8 oligos per gene model) A Kohler & F Martin

16. Secretome of Laccaria bicolor As a result of bio-informatic and array analyses of the L. bicolor genome, ~50 genes have been recognized that encode proteins (SSP) sharing features with fungal effectors secreted by pathogenic fungi into the host cells. The upregulation of these secreted small proteins in symbiotic tissues suggests that the symbiont releases products capable of manipulating the host. CFEM HESP148 SSPs

17. Laccaria: a saprophytic fungus... © INRA

18. Metabolic Zonation/ Physiological compartmentation in a series of hyphal networks Tunlid et al. (2005) New Phytologist, in press Early colonization: Symbiosis gene sets Hyphal webs on organic matter: Saprotrophism gene sets © Tunlid et al.

19. Genome evolution related to symbiotic lifestyle? Are there symbiosis gene families specifically amplified in Laccaria? Yes.. Mycorrhiza-induced Secreted Small cys-rich Proteins,. Mycorrhiza-induced Sensor Proteins (WD40, NACHT),. Receptors, GPCRs, GTPases Are genes required for litter degradation detected in Laccaria? Yes. Most genes required for saprotrophic growth in decaying leaf litter are found in Laccaria, but low cellulolytic ability. The latter will prevent it from degrading its host cells and becoming pathogenic. Janus Laccaria: a Janus-faced fungus... © INRA

20. Goals. Comparative genomics of ascomycetous and basidiomycetous ectomycorrhizal fungi. Identification of symbiosis factors. Molecular analysis of the fruiting body formation. Identification of flavors biosynthesis pathway. Identification of sex genes. Neutral and functional markers for population genetics Sur le sentier du Diamant noir …

21. F Martin, P Wincker, P Bonfante, S Ottonello, P Rouzé, G Chevalier, MH Lebrun CNS Génoscope Sequencing Center INRA - Torino & Parma Universities - VIB Tuber Genome Annotation Network Sequencing of the Genome of the Perigord Black Truffle Tuber melanosporum Current status of the project (November 2007) : - - 42,000 annotated ESTs of mycelium and fruiting body (see TuberDB) - - Genome sequencing: 8X assembly - 120 Mb - - - Annotation on going: FL cDNA training set, Transposon identification & masking

22. the Poplar Rust Melampsora larici-populina

23. F Martin, S Duplessis, R. Hamelin, P Rouzé, S.P. DiFazio, G.K. Podila & G.A. Tuskan Joint Genome Institute INRA - VIB - Canadian Forest Service ORNL - Alabama University - West Virginia University Melampsora Genome Annotation Network Sequencing of the Genome of the Poplar Rust Melampsora larici-populina Short summary of the 8X assembly (January 24, 2007) : - - Main genome read total:884,455 - - Main genome scaffold total:2682 - - Main genome contig total:14016 - - Main genome scaffold sequence total:120.0 MB - - Main genome contig sequence total:100.3 MB (16.4% gap) - - Main genome scaffold N/L50:64/469 KB - - Main genome contig N/L50:842/31.8 KB - - Main genome depth:7.32 ± 0.09 65,000 annotated ESTs of germlings & Populus-infected leaves (see MelampsoraDB)