Project description:Heterobasidion irregulare and H. occidentale are two closely related conifer root rot pathogens in the H. annosum sensu lato (s.l.) species complex. The two species H. irregulare and H. occidentale have different host preference with pine and non-pine tree species favored, respectively. The comparison of transcriptomes of H. irregulare and H. occidentale growing in Norway spruce bark, a susceptible host non-native to North America, showed large differences in gene expression. Heterobasidion irregulare induced more genes involved in detoxification of host compounds and in production of secondary metabolites, while the transcriptome induced in H. occidentale was more oriented towards carbohydrate degradation. Along with their separated evolutionary history, the difference might be driven by their host preferences as indicated by the differentially expressed genes enriched in particular Gene Ontology terms.
Project description:Estimating the fitness effect of deleterious mutations during the two phases of the life cycle: a new method applied to the root-rot fungus Heterobasidion parviporum
Project description:Transcript profiles of Heterobasion irregulare from different tissues and mycelium grown on different substrates were analyzed. The array probes were designed from gene models taken from the Joint Genome Institute (JGI, department of energy) Heterobasidion annosum genome sequence version 1. One aim of this study was to verify the expression of the automatically annotated gene models under various conditions. Another goal was to compare gene expression profiles from different tissues of Heterobasidion irregulare and from mycelium grown on liquid MMN medium, liquid medium amended with lignin or cellulose and on wood.
Project description:Root rot fungi of the Heterobasidion annosum complex are the most damaging pathogens in temperate forests, and the recently sequenced Heterobasidion irregulare genome revealed over 280 carbohydrate-active enzymes. Here, H. irregulare was grown on biomass, and the most abundant protein in the culture filtrate was identified as the only family 7 glycoside hydrolase in the genome, which consists of a single catalytic domain, lacking a linker and carbohydrate-binding module. The enzyme, HirCel7A, was characterized biochemically to determine the optimal conditions for activity. HirCel7A was crystallized and the structure, refined at 1.7 ? resolution, confirms that HirCel7A is a cellobiohydrolase rather than an endoglucanase, with a cellulose-binding tunnel that is more closed than Phanerochaete chrysosporium Cel7D and more open than Hypocrea jecorina Cel7A, suggesting intermediate enzyme properties. Molecular simulations were conducted to ascertain differences in enzyme-ligand interactions, ligand solvation, and loop flexibility between the family 7 glycoside hydrolase cellobiohydrolases from H. irregulare, H. jecorina, and P. chrysosporium. The structural comparisons and simulations suggest significant differences in enzyme-ligand interactions at the tunnel entrance in the -7 to -4 binding sites and suggest that a tyrosine residue at the tunnel entrance of HirCel7A may serve as an additional ligand-binding site. Additionally, the loops over the active site in H. jecorina Cel7A are more closed than loops in the other two enzymes, which has implications for the degree of processivity, endo-initiation, and substrate dissociation. Overall, this study highlights molecular level features important to understanding this biologically and industrially important family of glycoside hydrolases.
Project description:In conifer forests of Northern Europe, a pathogenic fungus Heterobasidion annosum attacks the roots of Scots pine and causes mortality. Trees with infection grow slower and produce less timber with reduced quality. Despite applied control methods, such as switching tree species to a non-host species, or stump treatment, root and butt rot continues to be a serious forest health problem. Disease resistance breeding is a less-applied control method which has potential to improve tree health. However, neither conifer genotypes with absolute resistance to Heterobasidion sp. nor robust selection markers for resistance breeding have been found. We studied the responses of various Scots pine genotypes to Heterobasidion annosum infection and mechanic damage in drained peatland. Stems and roots of mature naturally regenerated Scots pine trees growing in drained peatland were either artificially infected with H. annosum or wounded and inoculated with sterile inoculum. Untreated trees from the study sites served as controls. Responses of different Scots pine genotypes to pathogen infection as determined by lesion size were recorded from samples harvested four months after inoculation, and least susceptible and highly susceptible genotypes were selected from the study material. Analysis of terpenoids from both least susceptible and highly susceptible pine genotypes by gas chromatography coupled with mass spectrometry indicates that some monoterpenes and sesquiterpenes are differentially induced depending on the susceptibility level. Transcriptomic microarray analysis was therefore conducted with RNA from stems of the least susceptible and highly susceptible Scots pine genotypes. Gene expression data from cDNA microarray were analysed by comparisons between the treatments, and the genotypes with different resistance level. The aim of the study is to highlight transcripts specific to differing levels of susceptibility.
Project description:Lytic polysaccharide monooxygenases (LPMO) are important redox enzymes produced by microorganisms for the degradation of recalcitrant natural polysaccharides. Heterobasidion irregulare is a white-rot phytopathogenic fungus that causes wood decay in conifers. The genome of this fungus encodes 10 putative Auxiliary Activity family 9 (AA9) LPMOs. We describe the first biochemical characterization of H. irregulare LPMOs through heterologous expression of two CBM-containing LPMOs from this fungus (HiLPMO9H, HiLPMO9I) in Pichia pastoris. The oxidization preferences and substrate specificities of these two enzymes were determined. The two LPMOs were shown to cleave different carbohydrate components of plant cell walls. HiLPMO9H was active on cellulose and oxidized the substrate at the C1 carbon of the pyranose ring at ?-1,4-glycosidic linkages, whereas HiLPMO9I cleaved cellulose with strict oxidization at the C4 carbon of glucose unit at internal bonds, and also showed activity against glucomannan. We propose that the two LPMOs play different roles in the plant-cell-wall degrading system of H. irregulare for degradation of softwood and that the lignocellulose degradation mediated by this white-rot fungus may require collective efforts from multi-types of LPMOs.