Contribution à l'étude de la structure et du polymorphisme du génome du basidiomycète ectomycorhizien "Laccaria bicolor" (Maire) Orton et identification de QTLs de mycorhization chez les peupliers, "Populus trichocarpa Torr. & A. Gray ex Hook. et "Populus

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Contribution à l’étude de la structure et du polymorphisme du génome du basidiomycète ectomycorhizien ”Laccaria bicolor” (Maire) Orton et

identification de QTLs de mycorhization chez les peupliers, ”Populus trichocarpa Torr. & A. Gray ex

Hook. et ”Populus deltoides (Bartr.) Marsh

Jessy Labbé

To cite this version:

Jessy Labbé. Contribution à l’étude de la structure et du polymorphisme du génome du basidiomycète ectomycorhizien ”Laccaria bicolor” (Maire) Orton et identification de QTLs de mycorhization chez les peupliers, ”Populus trichocarpa Torr. & A. Gray ex Hook. et ”Populus deltoides (Bartr.) Marsh.

Sylviculture, foresterie. Université Henri Poincaré - Nancy 1, 2009. Français. �NNT : 2009NAN10079�.

�tel-01748313�

AVERTISSEMENT

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D'autre part, toute contrefaçon, plagiat, reproduction illicite encourt une poursuite pénale.

Contact : ddoc-theses-contact@univ-lorraine.fr

LIENS

Code de la Propriété Intellectuelle. articles L 122. 4

Code de la Propriété Intellectuelle. articles L 335.2- L 335.10

http://www.cfcopies.com/V2/leg/leg_droi.php

U.F.R. Sciences et Techniques Biologiques Ecole Doctorale Ressources Procédés Produits Environnement

UMR 1136 INRA/UHP Interactions arbres/Micro-organismes

Centre INRA de Nancy

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LETTERS

The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis

F. Martin¹, A. Aerts², D. Ahre´n³, A. Brun¹, E. G. J. Danchin⁴, F. Duchaussoy¹, J. Gibon¹, A. Kohler¹, E. Lindquist², V. Pereda¹, A. Salamov², H. J. Shapiro², J. Wuyts^1,5, D. Blaudez¹, M. Bue´e¹, P. Brokstein², B. Canba¨ck³, D. Cohen¹, P. E. Courty¹, P. M. Coutinho⁴, C. Delaruelle¹, J. C. Detter², A. Deveau¹, S. DiFazio⁶, S. Duplessis¹,

L. Fraissinet-Tachet⁸, E. Lucic¹, P. Frey-Klett¹, C. Fourrey¹, I. Feussner⁷, G. Gay⁸, J. Grimwood⁹, P. J. Hoegger¹⁰, P. Jain¹¹, S. Kilaru¹⁰, J. Labbe´¹, Y. C. Lin⁵, V. Legue´¹, F. Le Tacon¹, R. Marmeisse⁸, D. Melayah⁸, B. Montanini¹, M. Muratet¹¹, U. Nehls¹², H. Niculita-Hirzel¹³, M. P. Oudot-Le Secq¹, M. Peter^1,14, H. Quesneville¹⁵, B. Rajashekar³, M. Reich^1,10, N. Rouhier¹, J. Schmutz⁹, T. Yin¹⁶, M. Chalot¹, B. Henrissat⁴, U. Ku¨es¹⁰, S. Lucas², Y. Van de Peer⁵, G. K. Podila¹¹, A. Polle¹⁰, P. J. Pukkila¹⁷, P. M. Richardson², P. Rouze´^5,18, I. R. Sanders¹³, J. E. Stajich¹⁹, A. Tunlid³, G. Tuskan¹⁶& I. V. Grigoriev²

Mycorrhizal symbioses—the union of roots and soil fungi—are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants^1,2. Boreal, temperate and montane forests all depend on ectomycorrhizae¹. Identification of the prim- ary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored.

Here we report the genome sequence of the ectomycorrhizal basi- diomyceteLaccaria bicolor(Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase gen- ome assembly contains 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences.

We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown func- tion, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs prob- ably have a decisive role in the establishment of the symbiosis.

The unexpected observation that the genome of L. bicolorlacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall poly- saccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of theL. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availabi- lity of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts inter- act with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.

The 65-megabase genome ofLaccaria bicolor(Maire) P. D. Orton is the largest sequenced fungal genome published so far^3–7(Table 1).

Although no evidence for large-scale duplications was observed within theL. bicolor genome, tandem duplication occurred within multigene families (Supplementary Fig. 4). Transposable elements comprised a higher proportion (21%) than that identified in the other sequenced fungal genomes and may therefore account for the relatively large genome of L. bicolor(Supplementary Table 3).

Approximately 20,000 protein-coding genes were identified by com- bined gene predictions (Supplementary Information Section 2).

Expression of nearly 80% (,16,000) of the predicted genes was detected in free-living mycelium, ectomycorrhizal root tips or fruit- ing bodies (Supplementary Table 4) using NimbleGen custom- oligoarrays (Supplementary Information Section 9). Most genes are activated in almost all tissues, whereas other more specialized genes are only activated in some specific developmental stages, such as the free-living mycelium, ectomycorrhizae or the fruiting body (Supplementary Table 5).

Only 14,464L. bicolorproteins (70%) showed sequence similarity (BLASTX, cut-off e-value .0.001) to documented proteins. Most homologues were found in the sequenced basidiomycetesPhanero- chaete chrysosporium⁴, Cryptococcus neoformans⁵, Ustilago maydis⁶ andCoprinopsis cinerea⁷(Supplementary Table 6). The percentage of proteins found in multigene families was related to genome size and was the largest inL. bicolor(Fig. 2). This was mainly owing to the expansion of protein family size, but was also because of the larger number of protein families inL. bicolor compared with the other basidiomycetes (Supplementary Table 7). Expansion of protein family sizes in L. bicolorwas prominent in the lineage-specific multigene families. Marked gene family expansions occurred in those genes predicted to have roles in protein–protein interactions (for example, WD40-domain-containing proteins) and in signal transduction

1UMR 1136, INRA-Nancy Universite´, Interactions Arbres/Microorganismes, INRA-Nancy, 54280 Champenoux, France.²US DOE Joint Genome Institute, Walnut Creek, California 94598, USA.³Microbial Ecology, Lund University, SE-223 62 Lund, Sweden.⁴Architecture et Fonction des Macromole´cules Biologiques, UMR 6098 CNRS-Universite´s Aix-Marseille I

& II, 13288 Marseille Cedex 9, France.⁵Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, B-9052 Ghent, Belgium.

6Department of Biology, West Virginia University, Morgantown, West Virginia 26506, USA.⁷Department for Plant Biochemistry, Georg-August-Universita¨t Go¨ttingen, 37077 Go¨ttingen, Germany.⁸Universite´ Lyon 1, UMR CNRS - USC INRA d’Ecologie Microbienne, 69622 Villeurbanne, France.⁹Stanford Human Genome Center, Department of Genetics, Stanford University School of Medicine, 975 California Avenue, Palo Alto, California 94304, USA.¹⁰Institute of Forest Botany, Georg-August-Universita¨t, 37077 Go¨ttingen, Germany.

11Department of Biological Sciences, University of Alabama, Huntsville, Alabama 35899, USA.¹²Eberhard-Karls-Universita¨t, Physiologische Oekologie der Pflanzen, 72076 Tu¨bingen, Germany.¹³Department of Ecology & Evolution, University of Lausanne, 1015 Lausanne, Switzerland.¹⁴Swiss Federal Research Institute WSL, 8903 Birmensdorf, Switzerland.¹⁵Unite´ de Recherches en Ge´nomique-Info, INRA-Evry, 91034 E´vry Cedex, France.¹⁶Environmental Science Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA.

17Department of Biology, The University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.¹⁸Laboratoire Associe´ de l’INRA, Ghent University, B-9052 Gent, Belgium.

19Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102, USA.

Vol 452|6 March 2008|doi:10.1038/nature06556

88 ©2008Nature Publishing Group

mechanisms (Supplementary Table 7). Two new classes of GTPasea genes were found and may be candidates for the complex commun- ication that must occur between the mycobiont and its host plant during mycorrhizae establishment (Supplementary Table 8). Several transcripts coding for expanded and lineage-specific gene families were upregulated in symbiotic and fruiting body tissues, suggesting a role in tissue differentiation (Supplementary Tables 5 and 9).

In our analysis of annotated genes, in particular that of paralogous gene families, we highlighted processes that may be related to the biotrophic and saprotrophic lifestyles ofL. bicolor. Twelve predicted proteins showed a similarity to known haustoria-expressed secreted proteins of the basidiomycetous rustsUromyces fabae⁸andMelam- psora lini⁹, which are involved in pathogenesis (Supplementary Table 10). Out of the 2,931 proteins predicted to be secreted byL. bicolor, most (67%) cannot be ascribed a function, and 82% of these predicted proteins are specific toL. bicolor. Within this set, we found a large number of genes that encode cysteine-rich products that have a pre- dicted size of,300 amino acids. Of these 278 SSPs, 69% belong to multigene families, but only nine groups comprising a total of 33 SSPs co-localized in the genome (Supplementary Fig. 5). The structure of two of these clusters is shown in Supplementary Fig. 6. Other SSPs are scattered all over the genome, and we found no correlation between SSP and transposable element genome localization (Supplementary Fig. 5). Transcript profiling revealed that the expression of several SSP genes is specifically induced in the symbiotic interaction (Table 2 and Supplementary Fig. 10). Five of the 20 most highly upregulated fungal

transcripts in ectomycorrhizal root tips code for SSPs (Supplementary Table 5). These mycorrhiza-induced cysteine-rich SSPs (MISSPs) belong to L. bicolor-specific orphan gene families. Within the MISSPs, we found a family of secreted proteins with a CFEM domain (INTERPRO IPR014005) (Supplementary Figs 7 and 8), as previously identified in the plant pathogenic fungiM. lini⁹andMagnaporthea grisea¹⁰(Supplementary Table 10), and proteins with a gonadotropin (IPR0001545) or snake-toxin-like (SSF57302) domains related to the cysteine-knot domain. Expression of several SSPs was downregulated in ectomycorrhizal root tips (cluster E in Supplementary Fig. 10), suggesting a complex interplay between these secreted proteins in the symbiosis interaction.

The rich assortment of MISSPs may therefore act as effector pro- teins to manipulate host cell signalling or to suppress defence path- ways during infection, as suggested for pathogenic rusts^8,9, smuts⁶(U.

maydis) and Phytophthora¹¹ species. To have a role in symbiosis development, MISSPs should be expressed inL. bicolorhyphae col- onizing the root tips. To test this assertion, we determined the tissue distribution of the mycorrhiza-induced cysteine-rich SSP of 7 kDa (MISSP7) (JGI identification number 298595) showing the highest induction in ectomycorrhizal tips (Table 2 and Supplementary Table 5). Two peptides, one of which is located in the amino-terminal and the other in the carboxy-terminal part of the mature protein, were selected as antigens for the production of anti-MISSP7 antibodies. The selected peptides were not found in the deduced protein sequences of otherL.

bicolorgene models, nor in thePopulus trichocarpagenome¹². MISSP7

Table 1|Genome characteristics ofL. bicolorand other basidiomycetes

Genome characteristics L. bicolor C. cinerea⁷ P. chrysosporium⁴ C. neoformans⁵ U. maydis⁶

Strain S238N-H82 Okayama7#130 RP78 H99 521

Sequencing institution JGI Broad JGI Broad Broad

Genome assembly (Mb) 64.9 37.5 35.1 19.5 19.7

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Figure 1|Ectomycorrhizal symbiosis and the localization of the SSP MISSP7. a, Fruiting bodies ofL. bicolorcolonizing seedlings of Douglas fir (photograph courtesy of D. Vairelles).b, Laser-scanning confocal microscopy image of a transverse section ofPseudotsuga menziesii–L. bicolor ectomycorrhizae showing extramatrical mycelium (em), the mantle (m) and Hartig net hyphae (hn) between epidermal (ec), tannin (tc) and cortical (cc)

root cells. Scale, 10mm.c–f, Immunofluorescent localization of MISSP7.

Transverse (c,e) and longitudinal (d,f) sections ofP. trichocarpa–L. bicolor ectomycorrhizae. MISSP7 was detected in the hyphae of the mantle (m) and the Hartig net (hn) ensheathing epidermal cells (ec). Rectangles indand fshow the finger-like, labyrinthine hyphal system accumulating a large amount of MISSP7.e,f, Phase contrast images. Scale, 10mm.