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© 2006 - Cédric Pollet
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 StaffsGenoscope Production Sequencing StaffsPopulus Genome Annotation ConsortiumLaccaria Genome ConsortiumTuber Genome ConsortiumGlomus Genome ConsortiumPaxillus 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 StaffsGenoscope Production Sequencing StaffsPopulus Genome Annotation ConsortiumLaccaria Genome ConsortiumTuber Genome ConsortiumGlomus Genome ConsortiumPaxillus Genome Consortium INRA - AIP Séquençage
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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 GenomeF Martin, P Lammers, A Tunlid & G Tuskan
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Glomus
Melampsora
Laccaria
Populus
Tuber
Pseudomonas fluorescens
© F Martin - INRA
Détection d’un ensemble de SNP répartis sur l’ensemble du génome de Populus nigra
V. Jorge, I. Paolucci, A. Bresson, I. Bourgait, S. Schlub, P. Faivre-RampantAGPF, URGV
Objectif : disposer d’une large collection de SNP sur 2000 gènes pour des études d’association 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 l’espèce 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
Mycosphaerella
Alternaria
Leptosphaeria
Loculoascomycetes
Aspergillus
PodosporaChaetomiumFusarium
Magnaporthe
Eurotiomycetes
Sordariomycetes
Botrytis Sclerotinia
Leotiomycetes
Tuber Pezizomycetes
Saccharomyces CandidaOther yeasts
Schizosaccharomyces
Ustilago
Phanerochaete
Hemiascomycetes
Archeoascomycetes
Basidiomycetes
Yarrowia
Laccaria
Fungal Genomes 2007
Stagnospora
Puccinia Melampsora
Trichoderma
Coccidioides
Cryptococcus
Candida
Neurospora
Coprinopsis
saprotrophy
symbiosis
pathogenesis
Plant pathogens
Saprophytes
Plant Symbionts
Animal pathogens
Modified from MH Lebrun
© INRA
Agaricomycotina, Agaricales, Hydnangiaceae, Laccaria bicolor (Maire) P.D. Orton (bicoloured deceiver)
Metabolic Zonation/Physiological compartmentationin 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
© INRA
Basic features of the Laccaria genome ...
The Laccaria gene space
37% of the genome is coding sequence
19,128 putative 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
Expansion of protein families …
© INRA
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
The Laccaria secretome: a way to manipulate the host?
© INRA
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:
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
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
Laccaria: a saprophytic fungus ...
© INRA
Metabolic Zonation/Physiological compartmentationin 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.
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.
JanusJanus
Laccaria: a Janus-faced fungus ...
© INRA
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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 …
F Martin, P Wincker, P Bonfante, S Ottonello, P Rouzé, G Chevalier, MH Lebrun
CNS Génoscope Sequencing CenterINRA - Torino & Parma Universities - VIB
Tuber Genome Annotation Network
Sequencing of the Genome of the Perigord Black Truffle Tuber melanosporum
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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
the Poplar Rust Melampsora larici-populina
F Martin, S Duplessis, R. Hamelin, P Rouzé, S.P. DiFazio, G.K. Podila & G.A. Tuskan
Joint Genome InstituteINRA - VIB - Canadian Forest Service
ORNL - Alabama University - West Virginia UniversityMelampsora 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)