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.

De novo sequence assembly

A method for de novo assembly of a eukaryotic genome using Illumina, 454 and Sanger generated sequence data

Summary Grosmannia clavigera is a bark beetle‐vectored pine pathogen in the mountain pine beetle epidemic in western North America. Grosmannia clavigera colonizes pines despite the trees' massive oleoresin terpenoid defences. We are using a functional genomics approach to identify G. clavigera's mechanisms of adaptation to pine defences. We annotated the ABC transporters in the G. clavigera genome and generated RNA‐seq transcriptomes from G. clavigera grown with a range of terpenes. We functionally characterized GcABC‐G1, a pleiotropic drug resistance (PDR) transporter that was highly induced by terpenes, using qRT‐PCR, gene knock‐out and heterologous expression in yeast. Deleting GcABC‐G1 increased G. clavigera's sensitivity to monoterpenes and delayed development of symptoms in inoculated young lodgepole pine trees. Heterologous expression of GcABC‐G1 in yeast increased tolerance to monoterpenes. G. clavigera but not the deletion mutant, can use (+)‐limonene as a carbon source. Phylogenetic analysis placed GcABC‐G1 outside the ascomycete PDR transporter clades. G. clavigera appears to have evolved two mechanisms to survive and grow when exposed to monoterpenes: GcABC‐G1 controls monoterpene levels within the fungal cells and G. clavigera uses monoterpenes as a carbon source. This work has implications for understanding adaptation to host defences in an important forest insect–fungal system, and potentially for metabolic engineering of terpenoid production in yeast.

Sexual reproduction in fungi is regulated by the mating-type (MAT) locus where recombination is suppressed. We investigated the evolution of MAT loci in eight fungal species belonging to Grosmannia and Ophiostoma (Sordariomycetes, Ascomycota) that include conifer pathogens and beetle symbionts. The MAT1-2 idiomorph/allele was identified from the assembled and annotated Grosmannia clavigera genome, and the MAT locus is flanked by genes coding for cytoskeleton protein (SLA) and DNA lyase. The synteny of these genes is conserved and consistent with other members in Ascomycota. Using sequences from SLA and flanking regions, we characterized the MAT1-1 idiomorph from other isolates of G. clavigera and performed dotplot analysis between the two idiomorphs. Unexpectedly, the MAT1-2 idiomorph contains a truncated MAT1-1-1 gene upstream of the MAT1-2-1 gene that bears the high-mobility-group domain. The nucleotide and amino acid sequence of the truncated MAT1-1-1 gene is similar to its homologous copy in the MAT1-1 idiomorph in the opposite mating-type isolate, except that positive selection is acting on the truncated gene and the alpha(α)-box that encodes the transcription factor has been deleted. The MAT idiomorphs sharing identical gene organization were present in seven additional species in the Ophiostomatales, suggesting that the presence of truncated MAT1-1-1 gene is a general pattern in this order. We propose that an ancient unequal recombination event resulted in the ancestral MAT1-1-1 gene integrated into the MAT1-2 idiomorph and surviving as the truncated MAT1-1-1 genes. The α-box domain of MAT1-1-1 gene, located at the same MAT locus adjacent to the MAT1-2-1 gene, could have been removed by deletion after recombination due to mating signal interference. Our data confirmed a 1:1 MAT/sex ratio in two pathogen populations, and showed that all members of the Ophiostomatales studied here including those that were previously deemed asexual have the potential to reproduce sexually. This ability can potentially increase genetic variability and can enhance fitness in new, ecological niches.

Grosmannia clavigera is a fungal pathogen of pine forests in western North America and a symbiotic associate of two sister bark beetles: Dendroctonus ponderosae and D. jeffreyi. This fungus and its beetle associate D. ponderosae are expanding in large epidemics in western North America. Using the fungal genome sequence and gene annotations, we assessed whether fungal isolates from the two beetles inhabiting different species of pine in epidemic regions of western Canada and the USA, as well as in localized populations outside of the current epidemic, represent different genetic lineages. We characterized nucleotide variations in 67 genomic regions and selected 15 for the phylogenetic analysis. Using concordance of gene genealogies and distinct ecological characteristics, we identified two sibling phylogenetic species: Gc and Gs. Where the closely related Pinus ponderosa and P. jeffreyi are infested by localized populations of their respective beetles, Gc is present. In contrast, Gs is an exclusive associate of D. ponderosae mainly present on its primary host-tree P. contorta; however, in the current epidemic areas, it is also found in other pine species. These results suggest that the host-tree species and the beetle population dynamics may be important factors associated with the genetic divergence and diversity of fungal partners in the beetle-tree ecosystems. Gc represents the original G. clavigera holotype, and Gs should be described as a new species.