Project description:Gc, a Mountain pine beetle associated pathogen, can survive from highly abundant pine chemicals ( e.g terpenes) and use some unflavored compound’s as carbon source. using RNA_Seq, we analyzed the transcriptome of Gc when it grew on limonene, mannose oliver-oil, oleic acid as carbon source, as well as when it survived from high concentration of limonene or heptane. We profiled the expression of some interesting genes ( ABC transporters, P450s) potentially involved in the tree-pathogen interaction. An ABC-G group transporter gene (GcABC-G1) was one of the most highly induced genes and characterized as a mono-terpene specific efflux transporter with genetic and molecular tools. RNA- seq also indicated Gc utilize limonene and oleic acid through the same beta-oxidation pathway. However the degradation of limonene is more complex and multiple pathways contributed to the survival/utilization.
Project description:Gc, a Mountain pine beetle associated pathogen, can survive from highly abundant pine chemicals ( e.g terpenes) and use some unflavored compound’s as carbon source. using RNA_Seq, we analyzed the transcriptome of Gc when it grew on limonene, mannose oliver-oil, oleic acid as carbon source, as well as when it survived from high concentration of limonene or heptane. We profiled the expression of some interesting genes ( ABC transporters, P450s) potentially involved in the tree-pathogen interaction. An ABC-G group transporter gene (GcABC-G1) was one of the most highly induced genes and characterized as a mono-terpene specific efflux transporter with genetic and molecular tools. RNA- seq also indicated Gc utilize limonene and oleic acid through the same beta-oxidation pathway. However the degradation of limonene is more complex and multiple pathways contributed to the survival/utilization. mRNA was extracted from Gc mycelium under various conditions and cDNA libraries were generated for pair-end sequencing. The 70-100bp illumina sequence read was mapping to reference genome and RNA-Seq was carried out in CLC genomic work bench.
Project description:Wood and wood products can harbor microorganisms that can raise phytosanitary concerns in countries importing or exporting these products. To evaluate the efficacy of wood treatment on the survival of microorganisms of phytosanitary concern the method of choice is to grow microbes in petri dishes for subsequent identification. However, some plant pathogens are difficult or impossible to grow in axenic cultures. A molecular methodology capable of detecting living fungi and fungus-like organisms in situ can provide a solution. RNA represents the transcription of genes and can become rapidly unstable after cell death, providing a proxy measure of viability. We designed and used RNA-based molecular diagnostic assays targeting genes essential to vital processes and assessed their presence in wood colonized by fungi and oomycetes through reverse transcription and real-time polymerase chain reaction (PCR). A stability analysis was conducted by comparing the ratio of mRNA to gDNA over time following heat treatment of mycelial cultures of the Oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The real-time PCR results indicated that the DNA remained stable over a period of 10 days post treatment in heat-treated samples, whereas mRNA could not be detected after 24 hours for P. ramorum or 96 hours for G. clavigera. Therefore, this method provides a reliable way to evaluate the viability of these pathogens and offers a potential way to assess the effectiveness of existing and emerging wood treatments. This can have important phytosanitary impacts on assessing both timber and non-timber forest products of commercial value in international wood trade.
Project description: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.
Project description: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.