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Colette Breuil - One of the best experts on this subject based on the ideXlab platform.
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gene discovery for enzymes involved in limonene modification or utilization by the mountain pine beetle associated pathogen Grosmannia clavigera
Applied and Environmental Microbiology, 2014Co-Authors: Ye Wang, Joerg Bohlmann, Lina Madilao, Colette BreuilAbstract:To successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.
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comparative genomics of the pine pathogens and beetle symbionts in the genus Grosmannia
Molecular Biology and Evolution, 2014Co-Authors: Sepideh Massoumi Alamouti, Sajeet Haridas, Jörg Bohlmann, Gordon Robertson, Nicolas Feau, Colette BreuilAbstract:: Studies on beetle/tree fungal symbionts typically characterize the ecological and geographic distributions of the fungal populations. There is limited understanding of the genome-wide evolutionary processes that act within and between species as such fungi adapt to different environments, leading to physiological differences and reproductive isolation. Here, we assess genomic evidence for such evolutionary processes by extending our recent work on Grosmannia clavigera, which is vectored by the mountain pine beetle and jeffrey pine beetle. We report the genome sequences of an additional 11 G. clavigera (Gc) sensu lato strains from the two known sibling species, Grosmannia sp. (Gs) and Gc. The 12 fungal genomes are structurally similar, showing large-scale synteny within and between species. We identified 103,430 single-nucleotide variations that separated the Grosmannia strains into divergent Gs and Gc clades, and further divided each of these clades into two subclades, one of which may represent an additional species. Comparing variable genes between these lineages, we identified truncated genes and potential pseudogenes, as well as seven genes that show evidence of positive selection. As these variable genes are involved in secondary metabolism and in detoxifying or utilizing host-tree defense chemicals (e.g., polyketide synthases, oxidoreductases, and mono-oxygenases), their variants may reflect adaptation to the specific chemistries of the host trees Pinus contorta, P. ponderosa, and P. jeffreyi. This work provides a comprehensive resource for developing informative markers for landscape population genomics of these ecologically and economically important fungi, and an approach that could be extended to other beetle-tree-associated fungi.
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the genome and transcriptome of the pine saprophyte ophiostoma piceae and a comparison with the bark beetle associated pine pathogen Grosmannia clavigera
BMC Genomics, 2013Co-Authors: Sajeet Haridas, Rod Docking, Sepideh Massoumi Alamouti, Shaun D. Jackman, Jörg Bohlmann, Inanc Birol, Ye Wang, Gordon Robertson, Colette BreuilAbstract:Background Ophiostoma piceae is a wood-staining fungus that grows in the sapwood of conifer logs and lumber. We sequenced its genome and analyzed its transcriptomes under a range of growth conditions. A comparison with the genome and transcriptomes of the mountain pine beetle-associated pathogen Grosmannia clavigera highlights differences between a pathogen that colonizes and kills living pine trees and a saprophyte that colonizes wood and the inner bark of dead trees.
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a specialized abc efflux transporter gcabc g1 confers monoterpene resistance to Grosmannia clavigera a bark beetle associated fungal pathogen of pine trees
New Phytologist, 2013Co-Authors: Ye Wang, Jörg Bohlmann, Gordon Robertson, Scott Diguistini, Colette BreuilAbstract: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.
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the cytochromes p450 of Grosmannia clavigera genome organization phylogeny and expression in response to pine host chemicals
Fungal Genetics and Biology, 2013Co-Authors: Sajeet Haridas, Joerg Bohlmann, Colette BreuilAbstract:Grosmannia clavigera is a fungal associate of the mountain pine beetle (Dendroctonus ponderosae) and a pathogen of lodgepole pine (Pinus contorta) that must overcome terpenoid oleoresin and phenolic defenses of host trees. G. clavigera responds to monoterpene influx with complementary mechanisms that include export and the use of these compounds as a carbon source. Cytochromes P450 (CYPs) may also be involved in the metabolism of host defense compounds. We have identified and phylogenetically classified G. clavigera CYPs (CYPome). We show that although the G. clavigera CYPome has contracted in evolution, certain CYP families have expanded by duplication. We analyzed RNA-seq data for CYP expression following treatment with terpenes and pine phloem extracts to identify CYPs potentially involved in detoxification of these pine defense compounds. We also used transcriptome analysis of G. clavigera grown on monoterpenes, triglycerides or oleic acid as a carbon source to identify up-regulated CYPs that may be involved in the utilization of these compounds to support fungal growth. Finally, we identify secondary metabolite biosynthetic gene clusters that contain CYPs, and CYPs in clusters that may be involved in conversion of host chemicals.
Scott Diguistini - One of the best experts on this subject based on the ideXlab platform.
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unequal recombination and evolution of the mating type mat loci in the pathogenic fungus Grosmannia clavigera and relatives
G3: Genes Genomes Genetics, 2013Co-Authors: Clement K M Tsui, Richard C Hamelin, Jörg Bohlmann, Ye Wang, Nicolas Feau, Scott Diguistini, Braham DhillonAbstract: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.
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a specialized abc efflux transporter gcabc g1 confers monoterpene resistance to Grosmannia clavigera a bark beetle associated fungal pathogen of pine trees
New Phytologist, 2013Co-Authors: Ye Wang, Jörg Bohlmann, Gordon Robertson, Scott Diguistini, Colette BreuilAbstract: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.
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A specialized ABC efflux transporter GcABC‐G1 confers monoterpene resistance to Grosmannia clavigera, a bark beetle‐associated fungal pathogen of pine trees
New Phytologist, 2012Co-Authors: Ye Wang, Jörg Bohlmann, Gordon Robertson, Scott Diguistini, Colette BreuilAbstract: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.
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gene genealogies reveal cryptic species and host preferences for the pine fungal pathogen Grosmannia clavigera
Molecular Ecology, 2011Co-Authors: Sepideh Massoumi Alamouti, Richard C Hamelin, Jörg Bohlmann, Nicolas Feau, Scott Diguistini, Vincent Wang, Colette BreuilAbstract: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.
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genome and transcriptome analyses of the mountain pine beetle fungal symbiont Grosmannia clavigera a lodgepole pine pathogen
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Scott Diguistini, Ye Wang, Nicolas Feau, Nancy Y Liao, Simon K Chan, Uljana Hesseorce, Greg Taylor, Philippe Tanguay, Bernard Henrissat, Sepideh Massoumi AlamoutiAbstract: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.
Ye Wang - One of the best experts on this subject based on the ideXlab platform.
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gene discovery for enzymes involved in limonene modification or utilization by the mountain pine beetle associated pathogen Grosmannia clavigera
Applied and Environmental Microbiology, 2014Co-Authors: Ye Wang, Joerg Bohlmann, Lina Madilao, Colette BreuilAbstract:To successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.
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the genome and transcriptome of the pine saprophyte ophiostoma piceae and a comparison with the bark beetle associated pine pathogen Grosmannia clavigera
BMC Genomics, 2013Co-Authors: Sajeet Haridas, Rod Docking, Sepideh Massoumi Alamouti, Shaun D. Jackman, Jörg Bohlmann, Inanc Birol, Ye Wang, Gordon Robertson, Colette BreuilAbstract:Background Ophiostoma piceae is a wood-staining fungus that grows in the sapwood of conifer logs and lumber. We sequenced its genome and analyzed its transcriptomes under a range of growth conditions. A comparison with the genome and transcriptomes of the mountain pine beetle-associated pathogen Grosmannia clavigera highlights differences between a pathogen that colonizes and kills living pine trees and a saprophyte that colonizes wood and the inner bark of dead trees.
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unequal recombination and evolution of the mating type mat loci in the pathogenic fungus Grosmannia clavigera and relatives
G3: Genes Genomes Genetics, 2013Co-Authors: Clement K M Tsui, Richard C Hamelin, Jörg Bohlmann, Ye Wang, Nicolas Feau, Scott Diguistini, Braham DhillonAbstract: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.
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a specialized abc efflux transporter gcabc g1 confers monoterpene resistance to Grosmannia clavigera a bark beetle associated fungal pathogen of pine trees
New Phytologist, 2013Co-Authors: Ye Wang, Jörg Bohlmann, Gordon Robertson, Scott Diguistini, Colette BreuilAbstract: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.
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A specialized ABC efflux transporter GcABC‐G1 confers monoterpene resistance to Grosmannia clavigera, a bark beetle‐associated fungal pathogen of pine trees
New Phytologist, 2012Co-Authors: Ye Wang, Jörg Bohlmann, Gordon Robertson, Scott Diguistini, Colette BreuilAbstract: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.
Michael J. Wingfield - One of the best experts on this subject based on the ideXlab platform.
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phylogenetic re evaluation of the Grosmannia penicillata complex ascomycota ophiostomatales with the description of five new species from china and usa
Fungal Biology, 2020Co-Authors: Michael J. Wingfield, Xudong Zhou, Wilhelm Z De BeerAbstract:The Grosmannia penicillata complex (Ophiostomatales, Ascomycota) is one of the major species complexes in Leptographium sensu lato. Most of these are wood staining fungi associated with conifer-infesting bark beetles, and the complex encompasses the type species of the genus Grosmannia. Yet the phylogenetic relationships of species within the complex is unresolved. The aim of this study was to re-evaluate the circumscriptions of all known species in the G. penicillata complex, as well as isolates resembling G. penicillata obtained from a recent survey in China. Phylogenetic analyses of four gene regions: Internal transcribed spacer 2 and large subunit (ITS2-LSU), beta-tubulin (TUB), calmodulin (CAL), and translation elongation factor 1 alpha (TEF-1α) resolved the relationships of 15 species, including four new species (Grosmannia xianmiense sp nov., Grosmannia purpurea sp. nov., Grosmannia crassifolia sp. nov. and Grosmannia maixiuense sp. nov.), from China. Some isolates from pine in the USA that had previously been identified as Grosmannia abietina, represented a distinct taxon that is described here as Grosmannia xeno-abietina sp. nov.
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Mating type markers reveal high levels of heterothallism in Leptographium sensu lato.
Fungal Biology, 2016Co-Authors: Tuan A. Duong, Z. Wilhelm Beer, Brenda D Wingfield, Michael J. WingfieldAbstract:Species of Leptographium sensu lato are sap-stain fungi vectored by bark beetles and some species cause or are associated with tree diseases. Sexual states have been reported for more than 30 species in this group and these have been treated in the sexual genus Grosmannia. No sexual state is known for at least 59 additional species and these reside in the genus Leptographium. The discovery of sexual states for species of Leptographium relies mainly on the presence of fruiting bodies on host tissue at the time of isolation and/or intensive laboratory mating studies, which commonly have low levels of success. We developed mating-type markers to study sexual compatibility of species in Leptographium sensu lato. Using these markers, it was possible to identify mating types for 42 species and to determine thallism in many species for the first time. Surprisingly, the results showed that heterothallic and putatively heterothallic species are abundant (39 out of 42 species) in Leptographium sensu lato, and only three species were confirmed to be homothallic. The mating type markers developed in this study will be useful for future studies concerning mating type and sexual compatibility of species in this genus.
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microsatellite and mating type markers reveal unexpected patterns of genetic diversity in the pine root infecting fungus Grosmannia alacris
Plant Pathology, 2015Co-Authors: Tuan A. Duong, Brenda D Wingfield, Lori G Eckhardt, Z.w. De Beer, Michael J. WingfieldAbstract:Grosmannia alacris is a fungus commonly associated with root-infesting bark beetles occurring on Pinus spp. The fun- gus has been recorded in South Africa, the USA, France, Portugal and Spain and importantly, has been associated with pine root diseases in South Africa and the USA. Nothing is known regarding the population genetics or origin of G. alacris, although its association with root-infesting beetles native to Europe suggests that it is an invasive alien in South Africa. In this study, microsatellite markers together with newly developed mating type markers were used to characterize a total of 170 isolates of G. alacris from South Africa and the USA. The results showed that the genotypic diversity of the South African population of G. alacris was very high when compared to the USA populations. Two mating types were also present in South African isolates and the MAT1-1/MAT1-2 ratio did not differ from 1:1 (v 2 = 1� 39, P = 0� 24). This suggests that sexual reproduction most probably occurs in the fungus in South Africa, although a sexual state has never been seen in nature. In contrast, the large collection of USA isolates harboured only a single mating type. The results suggest that multiple introductions, followed by random mating, have influenced the population structure in South Africa. In contrast, limited introductions of probably a single mating type (MAT1-2) may best explain the clonality of USA populations.
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Ophiostomatoid fungi including two new fungal species associated with pine root-feeding beetles in northern Spain
Antonie van Leeuwenhoek, 2014Co-Authors: Pedro Romón, Z. Wilhelm Beer, Brenda D Wingfield, Mercedes Fernández, Julio Diez, Michael J. WingfieldAbstract:Many bark beetles live in a symbiosis with ophiostomatoid fungi but very little is known regarding these fungi in Spain. In this study, we considered the fungi associated with nine bark beetle species and one weevil infesting two native tree species ( Pinus sylvestris and Pinus nigra ) and one non-native ( Pinus radiata ) in Cantabria (Northern Spain). This included examination of 239 bark beetles or their galleries. Isolations yielded a total of 110 cultures that included 11 fungal species (five species of Leptographium sensu lato including Leptographium absconditum sp. nov., five species of Ophiostoma sensu lato including Ophiostoma cantabriense sp. nov, and one species of Graphilbum ). The most commonly encountered fungal associates of the bark beetles were Grosmannia olivacea, Leptographium procerum , and Ophiostoma canum . The aggressiveness of the collected fungal species was evaluated using inoculations on two-year-old P. radiata seedlings. Leptographium wingfieldii , Leptographium guttulatum , and Ophiostoma ips were the only species capable of causing significant lesions.
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Microsatellite and mating type markers reveal unexpected patterns of genetic diversity in the pine root‐infecting fungus Grosmannia alacris
Plant Pathology, 2014Co-Authors: Tuan A. Duong, Brenda D Wingfield, Lori G Eckhardt, Z.w. De Beer, Michael J. WingfieldAbstract:Grosmannia alacris is a fungus commonly associated with root-infesting bark beetles occurring on Pinus spp. The fun- gus has been recorded in South Africa, the USA, France, Portugal and Spain and importantly, has been associated with pine root diseases in South Africa and the USA. Nothing is known regarding the population genetics or origin of G. alacris, although its association with root-infesting beetles native to Europe suggests that it is an invasive alien in South Africa. In this study, microsatellite markers together with newly developed mating type markers were used to characterize a total of 170 isolates of G. alacris from South Africa and the USA. The results showed that the genotypic diversity of the South African population of G. alacris was very high when compared to the USA populations. Two mating types were also present in South African isolates and the MAT1-1/MAT1-2 ratio did not differ from 1:1 (v 2 = 1� 39, P = 0� 24). This suggests that sexual reproduction most probably occurs in the fungus in South Africa, although a sexual state has never been seen in nature. In contrast, the large collection of USA isolates harboured only a single mating type. The results suggest that multiple introductions, followed by random mating, have influenced the population structure in South Africa. In contrast, limited introductions of probably a single mating type (MAT1-2) may best explain the clonality of USA populations.
Janice E. K. Cooke - One of the best experts on this subject based on the ideXlab platform.
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a novel application of rnase h2 dependent quantitative pcr for detection and quantification of Grosmannia clavigera a mountain pine beetle fungal symbiont in environmental samples
Tree Physiology, 2018Co-Authors: Chandra H Mcallister, Colleen E Fortier, Kate R St Onge, Bianca M Sacchi, Meaghan J Nawrot, Troy Locke, Janice E. K. CookeAbstract:Mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) is an economically and ecologically important pest of pine species in western North America. Mountain pine beetles form complex multipartite relationships with microbial partners, including the ophiostomoid fungi Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Ophiostoma montium (Rumbold) von Arx, Grosmannia aurea (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Leptographium longiclavatum (Lee, Kim, and Breuil) and Leptographium terebrantis (Barras and Perry). These fungi are vectored by MPB to new pine hosts, where the fungi overcome host defenses to grow into the sapwood. A tree’s relative susceptibility to these fungi is conventionally assessed by measuring lesions that develop in response to fungal inoculation. However, these lesions represent a symptom of infection, representing both fungal growth and tree defense capacity. In order to more objectively assess fungal virulence and host tree susceptibility in studies of host–pathogen interactions, a reliable, consistent, sensitive method is required to accurately identify and quantify MPB-associated fungal symbionts in planta. We have adapted RNase H2-dependent PCR, a technique originally designed for rare allele discrimination, to develop a novel RNase H2-dependent quantitative PCR (rh-qPCR) assay that shows greater specificity and sensitivity than previously published PCR-based methods to quantify MPB fungal symbionts in pine xylem and MPB whole beetles. Two sets of assay probes were designed: one that amplifies a broad range of ophiostomoid species, and a second that amplifies G. clavigera but not other MPB-associated ophiostomoid species. Using these primers to quantify G. clavigera in pine stems, we provide evidence that lesion length does not accurately reflect the extent of fungal colonization along the stem nor the quantity of fungal growth within this colonized portion of stem. The sensitivity, specificity, reproducibility, cost effectiveness and high-throughput potential of the rh-qPCR assay makes the technology suitable for identification and quantification of a wide array of pathogenic and beneficial microbes that form associations with plants and other organisms, even when the microbial partner is present in low abundance.
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differences in defence responses of pinus contorta and pinus banksiana to the mountain pine beetle fungal associate Grosmannia clavigera are affected by water deficit
Plant Cell and Environment, 2016Co-Authors: Adriana Arangovelez, Walid El Kayal, Inka Lusebrink, Charles Copeland, Irina L Zaharia, Janice E. K. CookeAbstract:: We tested the hypotheses that responses to the mountain pine beetle fungal associate Grosmannia clavigera will differ between the evolutionarily co-evolved host lodgepole pine (Pinus contorta var. latifolia) and the naive host jack pine (Pinus banksiana) and that these responses will be influenced by water availability. G. clavigera inoculation resulted in more rapid stem lesion development in lodgepole than in jack pine; water deficit delayed lesion development in both species. Decreased hydraulic conductivity was observed in inoculated lodgepole pine seedlings, likely because of tracheid occlusion by fungal hyphae and/or metabolite accumulation. Drought but not inoculation significantly impacted bark abscisic acid levels. Jasmonic and salicylic acid were implicated in local and systemic responses of both species to G. clavigera, with salicylic acid appearing to play a greater role in jack pine response to G. clavigera than lodgepole pine. Water deficit increased constitutive levels and/or attenuated induced responses to G. clavigera for several monoterpenes in lodgepole but not jack pine. Instead, inoculation of well-watered but not water deficit jack pine resulted in a greater number of xylem resin ducts. These findings reveal mechanisms underlying differences in G. clavigera-induced responses between lodgepole and jack pine hosts, and how water availability modulates these responses.
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influence of water deficit on the molecular responses of pinus contorta pinus banksiana mature trees to infection by the mountain pine beetle fungal associate Grosmannia clavigera
Tree Physiology, 2014Co-Authors: Adriana Arangovelez, Leonardo Galindo M Gonzalez, Miranda Meents, Walid El Kayal, Barry J Cooke, Jean Linsky, Inka Lusebrink, Janice E. K. CookeAbstract:Conifers exhibit a number of constitutive and induced mechanisms to defend against attack by pests and pathogens such as mountain pine beetle (Dendroctonus ponderosae Hopkins) and their fungal associates. Ecological studies have demonstrated that stressed trees are more susceptible to attack by mountain pine beetle than their healthy counterparts. In this study, we tested the hypothesis that water deficit affects constitutive and induced responses of mature lodgepole pine × jack pine hybrids (Pinus contorta Dougl. ex Loud. var. latifolia Engelm. ex S. Wats. × Pinus banksiana Lamb.) to inoculation with the mountain pine beetle fungal associate Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield. The degree of stress induced by the imposed water-deficit treatment was sufficient to reduce photosynthesis. Grosmannia clavigera-induced lesions exhibited significantly reduced dimensions in water-deficit trees relative to well-watered trees at 5 weeks after inoculation. Treatment-associated cellular-level changes in secondary phloem were also observed. Quantitative RT-PCR was used to analyze transcript abundance profiles of 18 genes belonging to four families classically associated with biotic and abiotic stress responses: aquaporins (AQPs), dehydration-responsive element binding (DREB), terpene synthases (TPSs) and chitinases (CHIs). Transcript abundance profiles of a TIP2 AQP and a TINY-like DREB decreased significantly in fungus-inoculated trees, but not in response to water deficit. One TPS, Pcb(+)-3-carene synthase, and the Class II CHIs PcbCHI2.1 and PcbCHI2.2 showed increased expression under water-deficit conditions in the absence of fungal inoculation, while another TPS, Pcb(E)-β-farnesene synthase-like, and two CHIs, PcbCHI1.1 and PcbCHI4.1, showed attenuated expression under water-deficit conditions in the presence of fungal inoculation. The effects were observed both locally and systemically. These results demonstrate that both constitutive and induced carbon- and nitrogen-based defenses are affected by water deficit, suggesting potential consequences for mountain pine beetle dynamics, particularly in novel environments.
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Population structure of mountain pine beetle symbiont Leptographium longiclavatum and the implication on the multipartite beetle-fungi relationships.
PLOS ONE, 2014Co-Authors: Clement K M Tsui, Adrianne V Rice, Janice E. K. Cooke, Lina Farfan, Yousry A. El-kassaby, Richard C HamelinAbstract:Over 18 million ha of forests have been destroyed in the past decade in Canada by the mountain pine beetle (MPB) and its fungal symbionts. Understanding their population dynamics is critical to improving modeling of beetle epidemics and providing potential clues to predict population expansion. Leptographium longiclavatum and Grosmannia clavigera are fungal symbionts of MPB that aid the beetle to colonize and kill their pine hosts. We investigated the genetic structure and demographic expansion of L. longiclavatum in populations established within the historic distribution range and in the newly colonized regions. We identified three genetic clusters/populations that coincide with independent geographic locations. The genetic profiles of the recently established populations in northern British Columbia (BC) and Alberta suggest that they originated from central and southern BC. Approximate Bayesian Computation supports the scenario that this recent expansion represents an admixture of individuals originating from BC and the Rocky Mountains. Highly significant correlations were found among genetic distance matrices of L. longiclavatum, G. clavigera, and MPB. This highlights the concordance of demographic processes in these interacting organisms sharing a highly specialized niche and supports the hypothesis of long-term multipartite beetle-fungus co-evolutionary history and mutualistic relationships.
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Influence of water deficit on the molecular responses of Pinus contorta × Pinus banksiana mature trees to infection by the mountain pine beetle fungal associate, Grosmannia clavigera.
Tree Physiology, 2013Co-Authors: Adriana Arango-velez, Miranda Meents, Walid El Kayal, Barry J Cooke, Jean Linsky, Inka Lusebrink, Leonardo M. Galindo González, Janice E. K. CookeAbstract:Conifers exhibit a number of constitutive and induced mechanisms to defend against attack by pests and pathogens such as mountain pine beetle (Dendroctonus ponderosae Hopkins) and their fungal associates. Ecological studies have demonstrated that stressed trees are more susceptible to attack by mountain pine beetle than their healthy counterparts. In this study, we tested the hypothesis that water deficit affects constitutive and induced responses of mature lodgepole pine × jack pine hybrids (Pinus contorta Dougl. ex Loud. var. latifolia Engelm. ex S. Wats. × Pinus banksiana Lamb.) to inoculation with the mountain pine beetle fungal associate Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield. The degree of stress induced by the imposed water-deficit treatment was sufficient to reduce photosynthesis. Grosmannia clavigera-induced lesions exhibited significantly reduced dimensions in water-deficit trees relative to well-watered trees at 5 weeks after inoculation. Treatment-associated cellular-level changes in secondary phloem were also observed. Quantitative RT-PCR was used to analyze transcript abundance profiles of 18 genes belonging to four families classically associated with biotic and abiotic stress responses: aquaporins (AQPs), dehydration-responsive element binding (DREB), terpene synthases (TPSs) and chitinases (CHIs). Transcript abundance profiles of a TIP2 AQP and a TINY-like DREB decreased significantly in fungus-inoculated trees, but not in response to water deficit. One TPS, Pcb(+)-3-carene synthase, and the Class II CHIs PcbCHI2.1 and PcbCHI2.2 showed increased expression under water-deficit conditions in the absence of fungal inoculation, while another TPS, Pcb(E)-β-farnesene synthase-like, and two CHIs, PcbCHI1.1 and PcbCHI4.1, showed attenuated expression under water-deficit conditions in the presence of fungal inoculation. The effects were observed both locally and systemically. These results demonstrate that both constitutive and induced carbon- and nitrogen-based defenses are affected by water deficit, suggesting potential consequences for mountain pine beetle dynamics, particularly in novel environments.
