Analysis of ectomycorrhiza induced gene expression of selected poplar genes expressed in poplar fine roots

Abstract

Ectomycorrhiza is the symbiotic interacting between fungal hyphae and plant roots. The fungus provides the plant with nutrients mobilized from soil and the plant delivers carbohydrates from photosynthesis in exchange. Former studies showed an up-regulation of genes coding for the transcription factor dehydration responsive element binding factor 1 (DREB1) and for one facilitator of the gene family sugar will be eventually exported transporter (SWEET1) in the model organism poplar. While SWEET1 was shown to be involved as glucose transporter in diverse associations like pathogenic interactions, rhizobia nodules and arbuscular mycorrhiza, no link between DREB1 and such biotic interactions is reported. The Arabidopsis DREB1 homolog was shown to be involved in the regulation process of abscisic acid signaling and glucose related pathways. Aim of the thesis was the in vivo analysis of promoter fragments of the mentioned genes. For analysis promoter reporter constructs should be expressed in transgenic composite poplar. To distinguish between transgenic and non-transgenic roots, firstly a second, visual marker under control of a constitutive promoter should be integrated into the plant transformation vector. This step was necessary, since no classical selection procedure can be performed during composite plant generation leading to transgenic and non-transgenic roots emerging from the transformed shoot. A nuclear targeted Td-Tomato under control of the nopaline synthase (NOS) promoter turned out to give clear signals in poplar roots and was chosen as visual selection marker. Furthermore different binary vectors were tested for their transformation efficiency in composite poplar. The pCAMBIA-based vector pCXUN showed compared to pBi121 and the p-Green-based vector pPLV significantly increased transformation efficiency. To monitor expression of the promoter of interest, nuclear targeted super yellow fluorescence protein (sYFP) and double green fluorescence protein (dGFP) were analyzed. DGFP showed higher signal intensity in poplar roots and distinct localization to the nucleus in comparison to sYFP and was therefore used as marker gene in the final binary vector. Furthermore a transient expression system for poplar leaves was established, to analyze the activity of ectomycorrhiza induced promoters for their root specificity. Different techniques were tested and the infiltration of the fragile poplar leaves turned out to be most successful. The transient expression in poplar turned out to be not as robust as the infiltration of model organism N. benthamiana, but it enables a fast, transient expression in poplar. The newly composed vector pCXUN04NOS was used to analyze promoter fragments of DREB1 (3.2 kb) and SWEET1 (3.4 kb) for ectomycorrhiza dependent expression localization in composite poplar and expression in leaf tissue. To compare expression in mycorrhized and non-mycorrhized roots, composite plants were mycorrhized using the ectomycorrhiza fungi Pisolithus microcarpus and Amanita muscaria. The generated results stand in contrasted to previous expression data, since no ectomycorrhiza induced expression could be detected. Furthermore both fragments showed expression in leaf tissue of N. benthamiana and P. tremula x alba. These results indicate that the investigated promoter fragments did not contain all cis-elements important for ectomycorrhiza specific expression.