Project description:Trees are able to colonize, establish and survive in a wide range of soils through associations with ectomycorrhizal (EcM) fungi. Proper functioning of EcM fungi implies the differentiation of structures within the fungal colony. A symbiotic structure is dedicated to nutrient exchange and the extramatricular mycelium explores soil for nutrients. Eventually, basidiocarps develop to assure last stages of sexual reproduction. The aim of this study is to understand how an EcM fungus uses its gene set to support functional differentiation and development of specialized morphological structures. We examined the transcriptomes of Laccaria bicolor under a series of experimental setups, including the growth with Populus tremula x alba at different developmental stages, basidiocarps and free-living mycelium, under various conditions of N, P and C supply. In particular, N supply induced global transcriptional changes, whereas responses to P supply seemed to be independent from it. Symbiosis development with poplar is characterized by transcriptional waves. Basidiocarp development shares transcriptional signatures with other basidiomycetes. Overlaps in transcriptional responses of L. bicolor hyphae to a host plant and N/C supply next to co-regulation of genes in basidiocarps and mature mycorrhiza were detected. Few genes are induced in a single condition only, but functional and morphological differentiation rather involves fine tuning of larger gene sets. Overall, this transcriptomic atlas builds a reference to study the function and stability of EcM symbiosis in distinct conditions using L. bicolor as a model and indicates both similarities and differences with other ectomycorrhizal fungi, allowing researchers to distinguish conserved processes such as basidiocarp development from nutrient homeostasis.
Project description:BackgroundThe publicly available Laccaria bicolor genome sequence has provided a considerable genomic resource allowing systematic identification of transposable elements (TEs) in this symbiotic ectomycorrhizal fungus. Using a TE-specific annotation pipeline we have characterized and analyzed TEs in the L. bicolor S238N-H82 genome.Methodology/principal findingsTEs occupy 24% of the 60 Mb L. bicolor genome and represent 25,787 full-length and partial copy elements distributed within 171 families. The most abundant elements were the Copia-like. TEs are not randomly distributed across the genome, but are tightly nested or clustered. The majority of TEs exhibits signs of ancient transposition except some intact copies of terminal inverted repeats (TIRS), long terminal repeats (LTRs) and a large retrotransposon derivative (LARD) element. There were three main periods of TE expansion in L. bicolor: the first from 57 to 10 Mya, the second from 5 to 1 Mya and the most recent from 0.5 Mya ago until now. LTR retrotransposons are closely related to retrotransposons found in another basidiomycete, Coprinopsis cinerea.ConclusionsThis analysis 1) represents an initial characterization of TEs in the L. bicolor genome, 2) contributes to improve genome annotation and a greater understanding of the role TEs played in genome organization and evolution and 3) provides a valuable resource for future research on the genome evolution within the Laccaria genus.
Project description:BACKGROUND: Symbiotic ectomycorrhizal associations of fungi with forest trees play important and economically significant roles in the nutrition, growth and health of boreal forest trees, as well as in nutrient cycling. The ecology and physiology of ectomycorrhizal associations with Pinus sp are very well documented but very little is known about the molecular mechanisms behind these mutualistic interactions with gymnosperms as compared to angiosperms. RESULTS: Using a micro-array approach, the relative abundance of 2109 EST transcripts during interaction of Pinus sylvestris roots with the ectomycorrhizal fungus was profiled. The results reveal significant differential expression of a total of 236 ESTs, 96 transcripts differentially abundant after 1 day of physical contact with the fungus, 134 transcripts after 5 days and only 6 after 15 days at early stages of mantle formation on emerging lateral roots. A subset of cell wall modification and stress related genes was further assessed by quantitative reverse transcription PCR at late stages of mycorrhizal development coinciding with Hartig net formation. The results reveal down regulation of gene transcripts involved in general defence mechanism (e.g. antimicrobial peptide) as well as those involved in cell wall modification (e.g. glycine rich protein, xyloglucan endo transglycosylase). CONCLUSION: This study constitutes the first attempt to characterize the transcriptome of the plant partner in the Pinus sylvestris - Laccaria bicolor model system. We identified 236 ESTs which are potentially important for molecular regulation of a functional symbiotic association in conifer host. The results highlight similarities with other studies based on angiosperm model systems, nevertheless some differences were found in the timing and spatial scale of gene regulation during ectomycorrhiza development in gymnosperms. The present study has identified a number of potentially important molecular events responsible for the initiation and regulation of biochemical, physiological and morphological changes during development of a fully functional symbiosis that are relevant for gymnosperm hosts.
Project description:Illumina technology was used to generate mRNA profiles of three stages of L. bicolor basidiocarps. Total RNA was extracted separately from cap and stipe.TruSeq mRNA Stranded libraries were constructed and and 2 x 100 bp sequenced using Illumina HiSeq 2000 at the Genotoul sequencing facilities (Toulouse, France). Raw reads were trimmed for low quality (quality score 0.05), Illumina adapters and sequences shorter than 15 nucleotides and aligned to the L. bicolor v2 reference transcripts available at the JGI database https://mycocosm.jgi.doe.gov/Lacbi2/Lacbi2.home.html using CLC Genomics Workbench v8.
Project description:BACKGROUND: Accurate structural annotation is important for prediction of function and required for in vitro approaches to characterize or validate the gene expression products. Despite significant efforts in the field, determination of the gene structure from genomic data alone is a challenging and inaccurate process. The ease of acquisition of transcriptomic sequence provides a direct route to identify expressed sequences and determine the correct gene structure. METHODOLOGY: We developed methods to utilize RNA-seq data to correct errors in the structural annotation and extend the boundaries of current gene models using assembly approaches. The methods were validated with a transcriptomic data set derived from the fungus Laccaria bicolor, which develops a mycorrhizal symbiotic association with the roots of many tree species. Our analysis focused on the subset of 1501 gene models that are differentially expressed in the free living vs. mycorrhizal transcriptome and are expected to be important elements related to carbon metabolism, membrane permeability and transport, and intracellular signaling. Of the set of 1501 gene models, 1439 (96%) successfully generated modified gene models in which all error flags were successfully resolved and the sequences aligned to the genomic sequence. The remaining 4% (62 gene models) either had deviations from transcriptomic data that could not be spanned or generated sequence that did not align to genomic sequence. The outcome of this process is a set of high confidence gene models that can be reliably used for experimental characterization of protein function. CONCLUSIONS: 69% of expressed mycorrhizal JGI "best" gene models deviated from the transcript sequence derived by this method. The transcriptomic sequence enabled correction of a majority of the structural inconsistencies and resulted in a set of validated models for 96% of the mycorrhizal genes. The method described here can be applied to improve gene structural annotation in other species, provided that there is a sequenced genome and a set of gene models.
Project description:The Transcriptome of different tissues and developmental stages of Laccaria bicolor S238N was analyzed. The array probes were designed from gene models taken from the Joint Genome institute Laccaria bicolor genome sequence version1. One aim of this study was to verify the expression of the automatically annotated gene models in various tissues and to use this transcriptional information to confirm, to correct or to reject gene models. Another goal was to identify tissue-specific gene expression, e.g. mycorrhiza up-regulated transcripts or fruiting body up-regulated transcripts, or treatment specific gene expression for further detailed analyses. Keywords: Tissue comparison
Project description:The roots of four week old Populus × canescens were treated with Laccaria bicolor for one, two, four and seven days. Leaves three, four and five were harvested, pooled and used for RNA extraction and sequencing. The RNA of three biological samples was pooled after extraction. The experiment was repeated three times.
Project description:Illumina technology was used to generate mRNA profiles of Laccaria bicolor mycelium grown under different nutrien conditions. Total RNA was extracted.TruSeq mRNA Stranded libraries were constructed and and sequenced (2 x 150 bp Illumina HiSeq3000) at the Genotoul sequencing facilities (Toulouse, France). Raw reads were trimmed for low quality (quality score 0.05), Illumina adapters and sequences shorter than 15 nucleotides and aligned to the L. bicolor v2 reference transcripts available at the JGI database https://mycocosm.jgi.doe.gov/Lacbi2/Lacbi2.home.html using CLC Genomics Workbench v8.
Project description:Illumina GAIIx technology was used to generate mRNA profiles from the ectomycorrhizal fungi Laccaria bicolor colonizing roots of Populus trichocarpa. Samples were taken after two, four and 12 weeks of contact in order to identify mycorrhiza-regulated transcripts. 37bp reads were generated and aligned to the Populus trichocarpa (http://www.phytozome.net/poplar.php) and the Laccaria bicolor (http://genome.jgi-psf.org/Lacbi2/Lacbi2.home.html) reference genomes using CLC Genomics Workbench 6. mRNA profiles from Populus trichocarpa roots colonized by Laccaria bicolor for two, four and 12 weeks as well as from control roots and free-living mycelium were generated by using one lane of 37 bp Illumina GAIIx sequencing per sample.