Our project ventures to investigate extracts and compounds from fungi, but also from plants, for biological activity. Compounds that are generated in response to plant-fungal interaction are of particular interest. To screen for activity, we need assay systems that are fast, easy, reliable and need only small volumes. We want to meet this challenge by a kind of hybrid between a chip and a so called biological reporter, a strategy that is borrowed from Synthetic Biology. Plants or plant cells are „reconstructed“ such, that one can „see“ easily, whether they are just activating their immunity or resteer growth.
To follow the activation of immunity, plant cells are equipped at the Botanical Institute of the KIT with a gene construct that will generate, upon activation of plant defence, a green fluoresccence that can then be measured. For this purpose, we use the transcription factor MYB14. This gene switch from grapevines is always produced, when the so called basal immunity is activated, a kind of broad-band defence that wards off numerous pathogens. We use a particularly effective version of this switch, which we had found during a preceding Interreg Project (BACCHUS) in a European Wild Grape (Vitis sylvestris), the ancestor of our cultivated grapes, which meanwhile has been shifted to the verge of exctinction. The control sequence of MYB14 is placed in front of a piece of DNA that encodes the Green Fluorescent Protein, a protein, originating from jellyfish and responsible for marine glow. Each time, when one of our test compounds activates basal immunity, the control sequence of MYB14 will be activated and the cell will produce the Green Fluorescent Protein, leading to a greenish glow, which we can not only observe, but also measure. Meanwhile we have generated the gene construct and already introduced it into tobacco BY-2 cells. In the next step, we have to „calibrate“ this reporter system. For this purpose, we will treat the engineered cells with different quantitities of flg22, a bacterial peptide that activates basal immunity. After this „calibration“, our reporter system will be integrated into the microfluidic chip, and then we can start to measure the effect of the fungal strains coming from the IBWF Kaiserslautern.
In order to detect the effect of a fungal or plant compound on plant growth, a measurement system must be developed. In this project, we will measure the growth capabilities and growth rates of Arabidopsis thaliana seeds, also called Thale cress. To do this, the seeds are sown individually in agar filled plastic tips and placed on the biochip mounted on a glass slide, supplied by the KIT - Institute of Microstructure Technology. Four days after germination, the growing root enters a channel bathed in a flow of nutritive medium with (test) or without (control) contact with a fungal strain supplied by the IBWF Kaiserslautern. Pictures are taken under a magnifying glass and analyzing images makes it possible to calculate the speed of growth. When a nutritive perfusate induces a blockage or an acceleration of growth compared to the control, the study will continue on a microscopic scale by the analysis of the microtubular cytoskeleton and actin of the root cells and by the detection of possible cell death events. To do this, biochips mounted this time on glass slides will be used with transgenic Arabidopsis plants in which one of the fluorescent markers (TUB6-GFP; TUA6-mcherry; fimbrin-GFP; lifeAct-RFP) - obtained at IBMP, Strasbourg - combine with cytoskeletal proteins to determine the correlations between growth changes and the behavior of intracellular components involved in growth. These in vivo analyzes will establish a dynamic of induced cellular effects. Knowing that cortical microtubules control the axiality of cell expansion while actin controls the polar flow of a growth hormone (auxin), any compound capable of inhibiting either of these polymers is a potential candidate such as bio-herbicide. These in vivo analyzes will therefore make it possible to identify the signaling cascades which is induced and which produces the symptoms observed.
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Reference
Duan D, Fischer S, Merz PR, Bogs J, Riemann M, Nick P (2016) An ancestral allele of grapevine transcription factor MYB14 promotes plant defence. J Exp Bot 67, 1795-1804
