Transcript profiling using ESTs and cDNA arrays as a tool to study functional diversity in ectomycorrhizal fungi

M. Peter(1), M. Chalot(2), F. Le Tacon(1), and F. Martin(1)
UMR INRA-UHP 1136 Interactions Arbres/Micro-organismes,
(1) Centre INRA de Nancy, 54280 Champenoux
(2) UHP-Nancy 1, BP239 - 54506 Vandoeuvre-lès-Nancy

Several thousand fungal species worldwide form ectomycorrhizas with host trees. What is the functional significance of this diversity? Do different ectomycorrhizal species and genotypes differ in their functional abilities and thus offer distinct benefits for the host plant? Studying ectomycorrhizal fungi at the level of their transcriptome may be the key to the answers of these questions.
Therefore, we have studied the gene expression of two different genotypes of the ectomycorrhizal basidiomycete Laccaria bicolor interacting with the same host plant. We used a non-sterile symbiotic system with greenhouse-grown ectomycorrhizal seedlings of Douglas fir (Pseudotsuga menziesii) inoculated with both L. bicolor genotypes. The cDNA array technology was applied to investigate the expression of a large number of genes simultaneously. The specific questions were: do the two strains with different genetic background show differential gene expression when associated with the same host plant and is it possible to detect it?
As a prerequisite for gene expression profiling using cdna arrays we have constructed cdna libraries from mycelium of one of the two l. Bicolor genotypes and sequenced as well as functionally annotated approximately 1500 cdna clones. This large-scale production of expressed sequence tags (ests) allowed to discover ~900 different genes expressed in this ectomycorhizal basidiomycete. Sixty % of all ests did not show any similarity to previously identified genes within the genbank databases. A comparison of this set of ests with ests of other basidiomycetes, such as pisolithus microcarpus, revealed that l. Bicolor shared only around 10% of common transcripts with these fungi. This might indicate both, that the variability of expressed genes in different fungi and fungal tissues is considerably high and that there is a lack of genomic information of basidiomycetous origin.
In order to study the gene expression in ectomycorrhizas of the two l. Bicolor genotypes, the 900 different genes were spotted at least 4x on nylon arrays. A small section of each ectomycorrhizal root tip of three replicate plants was genotyped by analysing the igs1 region of ribosomal dna with an automated sequencer. After identification, the remaining root tissues were pooled for each of the three replicate plants and used as probes for cdna array hybridizations. The array analyses revealed that most of the genes were expressed equally in ectomycorrhizas of the two genotypes. However, it was possible to detect a small set of genes, which showed differential gene expression in all three replicates. The function of most of them is unknown and they are, therefore interesting candidates to study in more detail. The combined genotype/transcriptomic approach is a promising tool to study functional diversity of ectomycorrhizal fungi under different environmental conditions in microcosms and in the field.

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