Genome and transcriptome analyses of the mountain pine beetle-fungal symbiont Grosmannia clavigera, a lodgepole pine pathogen

Abstract

In western North America, the current outbreak of the mountain pine beetle (MPB) and its microbial associates has destroyed wide areas of lodgepole pine forest, including more than 16 million hectares in British Columbia. Grosmannia clavigera (Gc), a critical component of the outbreak, is a symbiont of the MPB and a pathogen of pine trees. To better understand the interactions between Gc, MPB, and lodgepole pine hosts, we sequenced the ∼30-Mb Gc genome and assembled it into 18 supercontigs. We predict 8,314 protein-coding genes, and support the gene models with proteome, expressed sequence tag, and RNA-seq data. We establish that Gc is heterothallic, and report evidence for repeat-induced point mutation. We report insights, from genome and transcriptome analyses, into how Gc tolerates conifer-defense chemicals, including oleoresin terpenoids, as they colonize a host tree. RNA-seq data indicate that terpenoids induce a substantial antimicrobial stress in Gc, and suggest that the fungus may detoxify these chemicals by using them as a carbon source. Terpenoid treatment strongly activated a ∼100-kb region of the Gc genome that contains a set of genes that may be important for detoxification of these host-defense chemicals. This work is a major step toward understanding the biological interactions between the tripartite MPB/fungus/forest system.

Fig. 1.

Life cycle and infection process of MPB and associated microorganisms. (A) MPBs disperse during early summer; both sexes of MPB carry blue stain fungi. Beetles bore through bark, make their galleries in the phloem, and deposit eggs along the gallery walls. During this process they introduce Gc and other associated microorganisms into the host tree. Fungi, yeast, and bacteria begin colonizing host tree tissues. Wood-staining Grosmannia and Ophiostoma fungi penetrate the xylem. The larvae feed on phloem creating galleries at right angles to the main galleries, completing their development after the fourth instar. Larvae pupate within the excavated chambers, and pupae transform into adults during early summer. During feeding, the larvae and beetles accumulate microorganisms in their guts, on their exoskeletons and in specialized maxillary structures known as mycangia. This process ensures that symbiotic microorganisms are transmitted to the next host. Gc colonizes the sapwood rapidly and produces a blue/black melanin pigment. Fungal growth blocks water and nutrient flow in the sapwood and phloem contributing to tree mortality. (B) Representative phenotypes of Gc. Light micrographs of a-sexual stage characterized with mononematous (i) and synnematous (ii) conidiophores reproducing conidia. (iii) Light micrograph of sexual structure characterized by a spherical ascocarp oozing ascospores. (iv) Stereomicrograph of conidiophores that grow inside the MPB gallery and ascocarps (Inset) on the inner bark of lodgepole pine. (C) Phylogenetic tree showing the positioning of Gc within the pezizomycotina.

Fig. 2.

Gene expression cluster induced following terpenoid treatment. RNA-seq profiling reveals a cluster of coexpressed genes on supercontig GCSC_108. For complete details and the results of genome-wide mapping data, see SI Appendix, Fig. S6. From Top to Bottom: transposons detected using de novo and reference based methods represented by black bars along supercontig. Expression analysis results derived from comparison of control vs. treatment for the 36-h terpenoid-treated samples averaged in 50-kb windows across GCSC_108. (Enlargement) Log-transformed coverage data for the peak expression region indicates agreement between predicted gene models and RNA-seq data.