DENIS, TAGU1 & SIMON HAWKINS2.
1INRA - Nancy, MicrobiologieForestière, 54280 Champenoux, France. 2INRA -Orléans, Génétique et Amélioration des Arbres Forestiers, Ardon45160 Olivet, France.
Ectomycorrhizas consist of integrated fungal and root tissues resulting from the juxtaposition of different cell types each fulfilling different functions and thus expressing a different set of genes. The temporal regulation of gene expression in ectomycorrhizal development can be followed by Northern analysis. However, the extraction of RNA, which is an integral part of such analysis, necessarily results in a loss of spatial information concerning the expression of genes. In addition in the case of mycorrhizas, both fungal and plant RNA's are mixed. The use of in situ localization techniques, in contrast however, provides detailed information on the spatial expression patterns of genes, and may very likely contribute to a greater understanding of the biological role of different tissues, cells and gene products. In this paper we propose to explore the possibility of localizing gene expression in ectomycorrhizal cells. As an optimization of the numerous steps of the protocol, we will present the distribution of the abundant fungal rRNA in Eucalyptus globulus -- Pisolithus tinctorius ectomycorrhizas. We will focus on particular problems which may be associated with ectomycorrhizal tissues and, where possible, we will propose adaptations of standard protocols. The major troubleshootings will be discussed: i) histological processing of hydrophobic tissues, ii) background hybridization and iii) size resolution of the samples. This communication is partly taken from Tagu and Hawkins (1998 Spatial regulation of genes in ectomycorrhiza: mRNA in situ localization and ß-glucuronidase detection. In "Mycorrhizal Manual "Springer Lab Manual, Berlin Germany, 449-462).