Regulation of mycorrhiza induced genes: identification of cis elements

Abstract

Ectomycorrhiza is a mutualistic interaction between higher land plants and certain fungi. The fungal partner receives carbohydrates from its plant partner and in return provides usable nitrogen and other nutrients. Not much is known about the mechanisms of the formation of the mycorrhizal structure and which signals play a role in both partners recognising each other. However, a number of genes have been found to be differentially regulated depending on mycorrhization. RNAsequencing and microarray analyses in the research group of Nehls and others have shown that many genes in poplar roots are differentially regulated depending on mycorrhiza. The aim of this work was to identify mycorrhiza-dependent elements in the promoter sequences of two genes, Potrx6G1673 and Potrx6G0093. For this purpose, corresponding promoter fragments were amplified from genomic DNA and tested in transgenic poplars. Composite poplars were produced in which only the roots are transgenic, but not the shoots (Neb et al. 2017). In this work, different visual markers were tested for their suitability to visualise the corresponding promoter fragments in the roots in order to detect differential expression of these fragments. Fluorescent proteins were chosen as visual markers because they allow in vivo analyses. On the one hand, super yellow fluorescent protein (sYFP) was tested with different promoter fragments in poplar roots, but due to the small size of the fluorescent protein, the visualisation results were not satisfactory. These problems occurred with both sYFP with nuclear localisation sequence and sYFP with peroxisomal localisation sequence. Therefore, double green fluorescent protein (dGFP) was established as a fluorescent marker for visualising promoter fragment activity. In the course of the work, it became apparent that the originally selected binary vector for plant transformation, pGreen, was not suitable for answering the research question of this work, so the produced constructs were cloned into another binary plant transformation vector, pCXUN. This system of binary vector and dGFP as visual marker was established in various experiments and subsequently successfully used to study the differential expression of corresponding poplar promoter fragments. Initial results indicate that the two promoters investigated appear to be subject to different regulatory mechanisms.