Samenvatting
The mutualistic symbiosis between ectomycorrhizal (ECM) fungi and a host plant is beneficial for both partners. The plant receives more nutrients and water and protection against pathogens, while the fungus gets carbohydrates in return. In the fungal metabolism, ROS play an important role in, for example, the development of mycorrhiza. Since Laccaria bicolor is the model organism of ECM fungi, the oxidative stress response was investigated in this fungus. Since the molecular mechanism of the ROS response is not yet fully understood in ECM fungi, it is important to develop tools to investigate this. A first objective was to create and optimize a construct that contains a genetically encoded fluorescent biosensor, that is H2O2-sensitive, for optimal fungal expression. In this thesis, the mitochondrial located biosensor Su9-roGFP-Tsa2ΔCR and the cytosolic located roGFP-Tsa2ΔCR were used to detect H2O2. The construct was obtained via several cloning techniques, such as PCR, restriction-ligation and Gibson assembly. A GPD intron was amplified and attached to both biosensors since this increases the efficiency of the GFP expression. First, pBGgHg was chosen as plasmid for the Agrobacterium-mediated transformation (AMT) procedure. Due to the lack of the sequence of this plasmid, several difficulties were encountered when creating the construct and trying to insert the biosensor into this plasmid. Therefore, a switch to the pPZP-P-T plasmid was made to use this plasmid for the transformation. The GPD intron – roGFP-Tsa2ΔCR construct was inserted into pPZP-P-T via Gibson assembly. After transformation into E. coli TOP10 competent cells, the sequence of the positive colonies were checked, and although the sequence in the beginning of the construct was correct, the sequence at the end of the biosensor showed lots of deletions and mutations. The GPD intron – Su9-roGFP-Tsa2ΔCR construct was inserted into pPZP-P-T by restriction-ligation. After transformation into E. coli TOP10 competent cells, one positive colony was obtained. The sequence was checked and the construct was inserted into pPZP-P-T in the correct orientation. Next, the plasmid was transformed into A. tumefaciens AGL1 and EHA105 competent cells. Also the empty plasmid was transformed into both A. tumefaciens strains to use as control. From each plasmid in each Agrobacterium strain, one positive colony was selected and used in the Laccaria transformation. Due to yeast contamination, the protocol was ended after the first selection step. A putative transcription factor in L. bicolor, and homolog of Yap1 in S. cerevisiae, is LbAP1. This gene might play a role in regulating the oxidative stress tolerance. Therefore, a second objective was to knockdown this gene to examine the in ROS metabolism-involved genes, that were regulated by this transcription factor. The method used to obtain this was RNA interference (RNAi). First, eight shRNA fragments were created, four containing an intron as spacer and four with a loop. The fragments were obtained with the help of Inverted Repeat Overlap PCR. Due to the obtained difficulties with the loop-containing fragments during the PCR reactions for the individual inverted repeats, these fragments were discarded. The intron-containing fragments were purified and inserted into pPZP-RNAi via Gibson assembly. The plasmid containing the RNAi fragments was then transformed into E. coli TOP10 competentcells. The colony PCR did not result in positive colonies that contained the construct of interest. A third objective was to find the dose of the selection marker needed for the AMT procedure. Therefore, an antibiotic range experiment was performed for both hygromycin and carboxin. The dose-response curve for each antibiotic was made by the drc package in R, based on the dry weight of the mycelium. Based on this curve, the antibiotic doses were selected for the use in AMT. For hygromycin, fungal growth was completely inhibited at 500 µg/ml and therefore, this concentration was chosen for the first selection step. In the case of carboxin, 10 µg/ml was selected. Since the pPZP-P-T plasmid was used for AMT, and since this plasmid contains a
carboxin resistance gene, 10 µg/ml was used in the first selection step.
| Datum prijs | 2024 |
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| Originele taal | English |