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Genome and Transcriptome Analyses of the Mountain Pine Beetle-Fungal Symbiont Grosmannia Clavigera, a Lodgepole Pine Pathogen

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Genome and Transcriptome Analyses of the Mountain Pine Beetle-Fungal Symbiont Grosmannia Clavigera, a Lodgepole Pine Pathogen Genome and transcriptome analyses of the mountain pine beetle-fungal symbiont Grosmannia clavigera, a lodgepole pine pathogen Scott DiGuistinia, Ye Wanga, Nancy Y. Liaob, Greg Taylorb, Philippe Tanguayc, Nicolas Feaud, Bernard Henrissate, Simon K. Chanb, Uljana Hesse-Orcea, Sepideh Massoumi Alamoutia, Clement K. M. Tsuif, Roderick T. Dockingb, Anthony Levasseurg, Sajeet Haridasa, Gordon Robertsonb, Inanc Birolb, Robert A. Holtb, Marco A. Marrab, Richard C. Hamelinc, Martin Hirstb, Steven J. M. Jonesb, Jörg Bohlmannf,h,1, and Colette Breuila,1 aDepartment of Wood Science, fDepartment of Forest Science, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; bBritish Columbia Cancer Agency Genome Sciences Centre, Vancouver, BC, Canada V5Z 4E6; cNatural Resources Canada, Ste-Foy, QC, Canada G1V 4C7; dUnité Mixte de Recherche 1202, Institut National de la Recherche Agronomique-Université Bordeaux I, Biodiversité, Gènes et Communautés, Institut National de la Recherche Agronomique Bordeaux-Aquitaine, 33612 Cestas Cedex, France; eArchitecture et Fonction des Macromolécules Biologiques, Unité Mixte de Recherche-6098, Centre National de la Recherche Scientifique, Universités Aix-Marseille I & II, 13288 Marseille cedex 9, France; gBiotechnologie des Champignons Filamenteux, Unité Mixte de Recherche-1161, Institut National de la Recherche, Universités de Provence et de la Méditerranée, 13288 Marseille cedex 09, France; and hMichael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z3 Edited by Rodney B. Croteau, Washington State University, Pullman, WA, and approved December 27, 2010 (received for review August 2, 2010) In western North America, the current outbreak of the mountain vectored fungi is symbiotic. The fungi benefit because beetles pine beetle (MPB) and its microbial associates has destroyed wide carry them through the tree bark into a new host’s nutrient-rich areas of lodgepole pine forest, including more than 16 million tissues. The benefits to the beetle and its progeny are less clear, hectares in British Columbia. Grosmannia clavigera (Gc), a critical but the fungi may make nutrients available and may detoxify component of the outbreak, is a symbiont of the MPB and a path- host-defense metabolites (5–7). Although both fungi and bark ogen of pine trees. To better understand the interactions between beetles must overcome physical and chemical host defenses to Gc, MPB, and lodgepole pine hosts, we sequenced the ∼30-Mb Gc become established in conifers, their relative contributions to fi genome and assembled it into 18 supercontigs. We predict 8,314 this process are poorly de ned. Toxic phenolics and oleoresin protein-coding genes, and support the gene models with pro- terpenoids are key chemical defense components in conifers (8, teome, expressed sequence tag, and RNA-seq data. We establish 9). In lodgepole pine, phenolics are stored in specialized poly- that Gc is heterothallic, and report evidence for repeat-induced phenolic parenchyma cells in the inner bark (phloem), and oleoresin monoterpenoids and diterpene resin acids are formed point mutation. We report insights, from genome and transcrip- and accumulate in resin ducts of the phloem and sapwood. When tome analyses, into how Gc tolerates conifer-defense chemicals, Gc is manually inoculated below the bark of seedlings or mature including oleoresin terpenoids, as they colonize a host tree. RNA- trees, as a single fungal inoculum point, it induces the formation seq data indicate that terpenoids induce a substantial antimicro- of a phloem lesion (i.e., a dark necrotic zone of tissue) that bial stress in Gc, and suggest that the fungus may detoxify these contains high concentrations of tree oleoresins and phenolics, chemicals by using them as a carbon source. Terpenoid treatment suggesting that the host prevents further fungal colonization. At ∼ strongly activated a 100-kb region of the Gc genome that con- higher inoculation densities, with inocula in multiple locations, tains a set of genes that may be important for detoxification of the fungus will also invade the sapwood adjacent to the lesions these host-defense chemicals. This work is a major step toward and block water transport to the crown of the tree (10). understanding the biological interactions between the tripartite Gc is specifically associated with the MPB, which colonizes only MPB/fungus/forest system. pine species, suggesting that both the vector and its fungal asso- ciates may have evolved specific metabolic pathways for over- next generation sequencing | monoterpene | carbohydrate active coming pine defenses. Although the virulence of Gc varies between enzymes | ABC transporter | forest genomics isolates (11), little systematic characterization has been performed on the genetic variation in Gc populations and on the relation of ark beetles and their fungal associates have inhabited conifer such variation to the differences in virulence between isolates. Bhosts since the Mesozoic era (1), and are the most eco- Identifying biochemical mechanisms by which Gc overcomes nomically and ecologically significant forest pests in the northern conifer defenses is a key part of understanding interactions be- hemisphere. The current outbreak of the mountain pine beetle tween this fungal pathogen and its host pine. To address this (MPB, Dendroctonus ponderosae) in western North America is knowledge gap, we first generated a draft genome sequence for the largest since the early 1900s. This beetle has killed an esti- Gc, primarily using next-generation sequencing data (12). Here, mated 630 million cubic meters (∼16.3 M hectares) of lodgepole we report the finished 29.8-Mb Gc genome sequence, 8,314 an- pine (Pinus contorta subsp. latifolia Engelm.) forest in British Columbia (www.for.gov.bc.ca/hfp/mountain_pine_beetle/). The MPB epidemic has bypassed the natural geographic barrier of Author contributions: S.D., S.J.M.J., J.B., and C.B. designed research; S.D., Y.W., N.Y.L., G.T., the Rocky Mountains and has the potential to spread eastward P.T., S.K.C., U.H.-O., S.M.A., and R.T.D. performed research; R.A.H., M.A.M., M.H., R.C.H., into the vast Canadian boreal pine forest. Climate change is and S.J.M.J. contributed new reagents/analytic tools; S.D., A.L., B.H., N.F., U.H.-O., C.K.M.T., thought to be a contributing factor to the current MPB epidemic, S.H., and I.B. analyzed data; and S.D., G.R., S.J.M.J., J.B., and C.B. wrote the paper. and the devastation of large areas of pine forest is anticipated to The authors declare no conflict of interest. have major consequences that include disturbing the global This article is a PNAS Direct Submission. balance of atmospheric carbon emission and sequestration (2). Freely available online through the PNAS open access option. Among the MPB-associated microbiota (3), the ascomycete Data deposition: The sequences reported in this paper have been deposited in NCBI Grosmannia clavigera (Gc) is a critical component of this large- GenBank as assembly and annotations accession ACXQ00000000. scale epidemic (Fig. 1). This pathogenic fungus can kill lodge- 1To whom correspondence may be addressed. E-mail: [email protected] or pole pine without the beetle when inoculated at a high density; [email protected]. however, the mechanisms by which the fungus kills trees are not This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fully characterized (4). The association between bark beetles and 1073/pnas.1011289108/-/DCSupplemental. 2504–2509 | PNAS | February 8, 2011 | vol. 108 | no. 6 www.pnas.org/cgi/doi/10.1073/pnas.1011289108 Downloaded by guest on September 27, 2021 notated protein coding sequences, initial annotation of protein repeat detection using RepeatScout (13). In total, 10.4% of the coding-sequence polymorphisms, proteins secreted in response finished genome was found to be composed of repeats or low to growth on wood, changes in the fungal transcriptome induced complexity sequences. Evidence for repeat-induced point muta- by exposure to lodgepole pine phloem extract (LPPE) or oleo- tion (RIP) was identified using RIPCAL (14), and was found resin terpenoids, and genes and pathways involved in the almost exclusively within transposable elements. After excluding modification, transport, and metabolism of conifer defense mitochondrial DNA, we predicted 8,314 protein-coding gene components. These resources and results provide a solid foun- models, accounting for 46% of the total genome length (SI Ap- dation to clarify the interaction of Gc with host-tree defenses. pendix, Dataset S1). The predicted gene models were supported and validated with EST, RNA-seq, and peptide sequences (Table Results 1). We annotated the translated set of sequences using public Genome Sequence and Protein Coding Annotations in G. clavigera. sequence databases and assigned initial functional descriptions Building on the previously published draft Gc genome sequence for ∼75% of the total predicted protein collection (SI Appendix, (12), we manually finished the genome assembly of Gc [kw1407; Dataset S1). National Center for Biotechnology Information (NCBI), Ge- nome PID: 39837] yielding 18 supercontigs with a total length of RNA-seq Validation of Gene Models and Identification of Protein 29.8 Mb (Table 1 and SI Appendix). Telomeric sequences sug- Coding Sequence Variations. To identify SNPs in the protein- gested that the supercontigs belonged to seven chromosomes. coding regions of the Gc genome and to provide additional gene We achieved 64× sequence coverage across 90% of the finished model support, we assessed the genome using RNA-seq read data genome sequence (SI Appendix, Figs. S1 and S2). We validated from a collection of seven additional Gc strains (SI Appendix, the assembly by aligning to it 99.4% of 7,169 unique expressed Table S1) (42 different culture-treatment combinations). For this sequence tag (EST) sequences (method described in ref. 12). We purpose we generated cDNA from polyA+ purified total RNA assembled the mitochondrial genome into a single ∼90-kb cir- and sequenced it using a paired-end read approach on the Illu- cularized sequence (SI Appendix, Fig.
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