Project description:To identify specific gene networks induced in host roots by C. geophilum, we inoculated seedlings of Scots pine simultaneously with C. geophilum and either Suillus granulatus or Rhizopogon roseolus, two common ECM fungi associated to pines. We then measured the differential expression of Scots pine genes in the respective mycorrhizas using oligoarrays.
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:To identify specific gene networks induced in host roots by C. geophilum, we inoculated seedlings of Scots pine simultaneously with C. geophilum and either Suillus granulatus or Rhizopogon roseolus, two common ECM fungi associated to pines. We then measured the differential expression of Scots pine genes in the respective mycorrhizas using oligoarrays. We performed 14 hybridizations (NimbleGen) with samples derived from Pinus sylvestris mycorrhiza with Cenococcum geophilum, Rhizopogon roseolus or Suillus granulatus (3 biological replicates each), as well as from non-mycorrhizal control roots (two replicates). Only the Pinus-derived sequences from the array were considered for this analysis. All samples were labeled with Cy3.
Project description:The meristem-associated endosymbiont M. extorquens DSM13060 significantly increases needle and root growth of Scots pine (Pinus sylvestris L.) seedlings without producing plant hormones, but by aggregating around host nuclei. Here we studied gene expression of the pine host induced by M. extorquens DSM13060 infection. We selected the time point of 90 days post-inoculation for our analysis based, because at this point, Methylorubrum extorquens DSM13060 has systemically colonized the pine seedlings, being found throughout tissues of roots and shoots.
Project description:Understanding of mechanisms of resistance of forest trees against microbial pathogens is an essential prerequisite for the development of sustainable forestry practices and for the improvement of commercially-grown trees via either conventional breeding or rational genetic engineering. We have studied the transcriptional response of Scots pine trees to Heterobasidion annosum infection under field conditions. By comparing responses of trees to wounding and to fungal inoculation we could identify a set of genes that were specifically responding to fungal infection. We have also investigated a contribution of Scots pine antimicrobial protein Sp-AMP2 to the host antimicrobial defense to evaluate the potential of Sp-AMP genes as molecular markers for resistance breeding.
Project description:Transcript profiles of H. annosum from different tissues and mycelium grown on different substrates and under different stresses were analyzed. The array probes were designed from gene models taken from the Joint Genome Institute (JGI, Department of Energy) H. irregulare genome sequence version 1. One aim of this study was to compare gene expression profiles of H. annosum grown on different substrates and under different biotic and abiotic stresses. We performed hybridizations (NimbleGen) with samples derived from H. annosum grown in either liquid Malt Extract Medium at 20°C (three biological replicates), under NaCl stress (three biological replicates), under CaCl2 stress (three biological replicates), at 8°C (three biological replicates), at 27°C (three biological replicates), saprotrophic growth on Scots pine sapwood (three biological replicates), bark (three biological replicates) or heartwood (three biological replicates). Additional hybridizations were performed with RNA from necrotrophic growth of Heterobasidion on Scots pine xylem (three biological replicates), necrotrophic growth on Scots pine phloem (three biological replicates), under nutrient starvation (three biological replicates) or under oxidative stress (three biological replicates). The Heterobasidion irregulare custom-exon expression array (4 x 72K) manufactured by Roche NimbleGen Systems Limited (Madison, WI) (http://www.nimblegen.com/products/exp/index.html) contained five independent, non-identical, 60-mer probes per gene model coding sequence. For 12,199 of the 12,299 annotated protein-coding gene models, probes could be designed. For 19 gene models, no probes could be generated, and 81 gene models shared all five probes with other gene models. Included in the array were 916 random 60-mer control probes and labelling controls. For 2032 randomly chosen gene models, technical duplicates were included on the array.
Project description:Comparative transcriptome analysis of early interaction events in Scots pine root tissues following challenge with a pathogenic, saprophytic or symbiotic fungus. Seedlings of P. sylvestris (19 days post germination) were transferred to wet, sterile filter paper on Petri-plates. Thereafter, the roots of the seedlings were inoculated with the mycelial homogenate of either Heterobasidion annosum (FP5, P-type) a pathogenic root rot fungus which attacks Norway spruce, Scots pine and broad leaf trees or Laccaria bicolor, an obligate ectomycorrhizal symbiont or Trichoderma aureoviride- an obligate saprotroph. Thereafter, incubated for 30 minutes, during which time some hyphae adhered to the roots. The inoculated seedlings (ten) were then transferred to another wet sterile filter paper placed on 1% water agar in Petri dishes. A second set of moist sterile filter paper was laid over the roots. The region of the Petri-dish containing the roots was covered with aluminium foil and the edges of the plate sealed with parafilm. The seedlings were then incubated for 24 hr under a photoperiod of 16h light at 20 ºC. Control seedlings were âinoculatedâ with sterile distilled water.
Project description:Comparative transcriptome analysis of early interaction events in Scots pine root tissues following challenge with a pathogenic, saprophytic or symbiotic fungus. Seedlings of P. sylvestris (19 days post germination) were transferred to wet, sterile filter paper on Petri-plates. Thereafter, the roots of the seedlings were inoculated with the mycelial homogenate of either Heterobasidion annosum (FP5, P-type) a pathogenic root rot fungus which attacks Norway spruce, Scots pine and broad leaf trees or Laccaria bicolor, an obligate ectomycorrhizal symbiont or Trichoderma aureoviride- an obligate saprotroph. Thereafter, incubated for 30 minutes, during which time some hyphae adhered to the roots. The inoculated seedlings (ten) were then transferred to another wet sterile filter paper placed on 1% water agar in Petri dishes. A second set of moist sterile filter paper was laid over the roots. The region of the Petri-dish containing the roots was covered with aluminium foil and the edges of the plate sealed with parafilm. The seedlings were then incubated for 24 hr under a photoperiod of 16h light at 20 ºC. Control seedlings were ‘inoculated’ with sterile distilled water. Keywords: other