Ectomycorrhiza Development : Investigation of Selected Ectomycorrhiza Induced Poplar Genes

Abstract

Mutualistic interaction such as ectomycorrhiza (ECM) is important for forest ecosystem function. Here two kingdoms, plant and fungi, form a symbiosis and exchanges nutrients and carbohydrates. A bottle neck in ectomycorrhizal research is the time demand for transgenic plant generation. Formation of so-called composite plants, where transgenic roots are formed on non-transgenic shoots, is an alternative strategy. An Agrobacterium rhizogenes-mediated root transformation protocol was developed in this work using axenic Populus tremula A tremuloides and P. tremula A alba cuttings. Out of four different A. rhizogenes strains, K599 was found to be the most suitable one. Roots of composite poplars were able to form ectomycorrhiza when inoculated with Amanita muscaria. By using real time quantitative PCR a comparative analysis of transcript levels was done for selected genes in mycorrhized and non-mycorrhized poplar fine roots. A total of 50 ectomycorrhiza-induced genes were chosen based on a genome wide microarray analysis (Nehls, unpublished). As the array oligomers were designed based on Populus trichocarpa genome but the array hybridization was performed using P. tremula x tremuloides cDNA, cross hybridization leading to misinterpretation is feasible. Therefore, the first step was to screen P. tremula/P. tremuloides datasets for the best matching homologs. After primer design and first qRT-PCR tests 14 candidate genes remained. Finally, expression analysis with several independent batches of poplar fine roots and mycorrhizas were obtained for six genes. Two genes, a transcription factor (TF- Potri.008G071100) belonging to the AP2/ERF superfamily and a potential glycosyltransferase (GT- Potri.007G095000) revealing the highest gene expression difference, were selected for further analysis. Fusion with super yellow fluorescent protein revealed a probable subcellular localization of Potri.007G095000 in nucleus and cytoplasm and Potri.008G071100 in nucleus. Following their establishment, composite poplars were used for promoter analysis of the selected genes. For this purpose, around 3 kb promoter fragments were amplified from genomic DNA of P. tremula x tremuloides, successively shortened from the 5' end and the resulting fragments were cloned in front of the coding sequence of a peroxisomal located yellow fluorescent protein. The longest promoter-reporter constructs were used for generation of composite poplars. No reliable ECM induced expression was found for the TF and the GT indicating the need for longer promoter fragments. Furthermore, as the best Arabidopsis TF homolog is known to be auto-regulated, the identification of potential cis-elements in the promoter region was planned. Therefore, protein overexpression in E. coli was initiated. However, all attempts of receiving a natively folded protein in soluble form were unsuccessful.