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Abstract Background Grosmannia clavigera is a bark beetle-vectored fungal pathogen of pines that causes wood discoloration and may kill trees by disrupting nutrient and water transport. Trees respond to attacks from beetles and associated fungi by releasing terpenoid and phenolic defense compounds. It is unclear which genes are important for G. clavigera's ability to overcome antifungal pine terpenoids and phenolics. Results We constructed seven cDNA libraries from eight G. clavigera isolates grown under various culture conditions, and Sanger sequenced the 5' and 3' ends of 25,000 cDNA clones, resulting in 44,288 high quality ESTs. The assembled dataset of unique transcripts (unigenes) consists of 6,265 contigs and 2,459 singletons that mapped to 6,467 locations on the G. clavigera reference genome, representing ~70% of the predicted G. clavigera genes. Although only 54% of the unigenes matched characterized proteins at the NCBI database, this dataset extensively covers major metabolic pathways, cellular processes, and genes necessary for response to environmental stimuli and genetic information processing. Furthermore, we identified genes expressed in spores prior to germination, and genes involved in response to treatment with lodgepole pine phloem extract (LPPE). Conclusions We provide a comprehensively annotated EST dataset for G. clavigera that represents a rich resource for gene characterization in this and other ophiostomatoid fungi. Genes expressed in response to LPPE treatment are indicative of fungal oxidative stress response. We identified two clusters of potentially functionally related genes responsive to LPPE treatment. Furthermore, we report a simple method for identifying contig misassemblies in de novo assembled EST collections caused by gene overlap on the genome.

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. [ABSTRACT FROM AUTHOR]

Grosmannia clavigera is a fungal pathogen associated with the mountain pine beetle ( Dendroctonus ponderosae) which is devastating large areas of western Canada’s conifer forests. This fungus also produces a dark melanin pigment that discolors pine sapwood. We have generated the draft genome of G. clavigera. However, functional characterization of genes identified in the genome sequence requires an efficient gene disruption method. In this work, we report a gene replacement strategy for G. clavigera using the Agrobacterium-mediated transformation in conjunction with linear or split-marker deletion cassettes. In addition, we used long flanking regions up to 3 kb from both sides of the targeted genes in our deletion cassettes. We assessed this gene disruption method with two genes from the melanin biosynthesis pathway that produce easily detectable white and red/brown mutant phenotypes: polyketide synthase and scytalone dehydratase. The approach yielded G. clavigera gene replacements with homologous recombination rates between 65 and 82%. For filamentous fungi, this is the first report showing that split-markers can be used with Agrobacterium-mediated transformation to generate appropriate mutants. This method can now be applied to efficiently identify genes involved in G. clavigera fungal pathogenicity and will facilitate understanding how the fungus overcomes the host defence system. [ABSTRACT FROM AUTHOR]

The article reports on a study of the differences in pathogenicity of Grosmannia clavigera, a blue-staining fungus associated with the mountain pine beetle, on young lodgepole pine in British Columbia. It made inoculations of Grosmannia clavigera fungi into young lodgepole pine trees. It founds out that Grosmannia clavigera isolates differed in terms of lesions, occluded larger areas, and sapwood moisture content.

Ophiostoma clavigerum is a destructive pathogen of lodgepole pine ( Pinus contorta) forests in western North America. It is therefore a relevant system for a genomics analysis of fungi vectored by bark beetles. To begin characterizing molecular interactions between the pathogen and its conifer host, we created an expressed sequence tag (EST) collection for O. clavigerum. Lodgepole pine sawdust and oleoresin media were selected to stimulate gene expression that would be specific to this host interaction. Over 6500 cDNA clones, derived from four normalized cDNA libraries, were single-pass sequenced from the 3′ end. After quality screening, we identified 5975 high-quality reads with an averagephred 20 of greater than 750 bp. Clustering and assembly of this high-quality EST set resulted in the identification of 2620 unique putative transcripts.blastx analysis revealed that only 67% of these unique transcripts could be matched to known or predicted protein sequences in public databases. Functional classification of these sequences provided initial insights into the transcriptome of O. clavigerum. Of particular interest, our ESTs represent an extensive collection of cytochrome P450 s, ATP-binding-cassette-type transporters and genes involved in 1,8-dihydroxynaphthalene-melanin biosynthesis. These results are discussed in the context of detoxification of conifer oleoresins and fungal pathogenesis. [ABSTRACT FROM AUTHOR]

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 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., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)