Plant researchers led by Dr. Nico Dissmeyer, head of the independent junior research group on Protein Recognition and Degradation, and scientists from Switzerland have published a new method for obtaining valuable proteins from plants. The technique was published in Plant Physiology under the motto "Phenotype on demand".
The method is based on an artificial gene fusion of a temperature-sensitive degron cassette (lt-degron) with a gene coding for a protein of interest. The scientists expressed the generated gene fusion in Arabidopsis thaliana plants and exposed those to different temperatures. An increased outside temperature led to the destabilization of the temperature-labile lt-degron, which was recognized as defective by the proteasome and degraded together with the fused protein of interest. If the ambient temperature is kept low, the stable fusion protein can accumulate.
In 2016, this method was already established by the group around Dr. Nico Dissmeyer. It included a lt-degron gene fusion with the DNA sequence of an regulator of trichome development, as the protein of interest. The fusion was introduced into an Arabidopsis mutant lacking trichomes. Lowering the ambient temperature to 13°C led to the accumulation of the stable fusion protein, which enabled the correct formation of trichomes. Cultivating the plants at 29°C led to a destabilized fusion protein, thus blocking trichome development and resulting in smooth, hairless plants.
The current method provides the possibility to produce even cytotoxic proteins via a temperature shift. To demonstrate this, the plant researchers fused the gene of the bacterial ribonuclease 'barnase', an unspecific and highly toxic RNAse, to the gene of the temperature-labile lt-degron. The fusion was expressed under a trichome-specific promoter in wildtype Arabidopsis plants, which normally develop regular plant hairs. At low temperatures, the cytotoxic protein fusion was stable. Therefore, trichome development was apparently disturbed by the functional barnase and hairless leaves were formed. When cultivated at elevated outdoor temperatures, the artificially introduced cytotoxin was degraded in the trichome cells. Consequently, the cytotoxic effect was switched off and newly formed leaves exhibited regular plant hairs again. By multiple temperature changes, this process could even be toggled several times in individual plants, so that new leaf formations first occurred without, in the meantime with and finally free of trichomes.
As certain plant species develop trichomes with glandular function, it could be useful to transfer the method from the model plant Arabidopsis to these species. These glandular trichomes form a closed cell system and store various metabolic products, such as essential oils and insect repellents, as well as toxic degradation products and substances such as heavy metals. Glandular trichomes occupy only a small part of the leaf, but can account for up to 15 % of the plant's dry weight with their accumulated substances. Plants with larger biomass and leaves than Arabidopsis, such as tobacco or tomato plants, are suitable, because the trichomes of these plants could be used very well to produce pharmaceutically valuable proteins. Numerous cytotoxic proteins are currently being investigated for their potential use in cancer therapy.
The biosynthesis of proteins in plants has numerous advantages over previous methods carried out in yeast and cell cultures: First, the application of the method is comparatively simple and production of the protein of interest can be adjusted via the temperature. Futhermore, the use of exogenous substances and inducible promoters is not necessary. This reduces the probability of an uneven protein production in the plant. In addition, the protein biosynthesis with plant-specific glycosylations and folding, which can only be achieved in eukaryotes, is conceivable. Moreover, the lt-degron fusion system could also be transferred to other multicellular organisms such as insects and higher animals. The method is also interesting as a potential tool in genetics and developmental biology, where targeted cell ablation remains still challenging.
Original publication:
Frederik Faden, Stefan Mielke & Nico Dissmeyer, Modulating protein stability to switch toxic protein function on and off in living cells. Plant Physiology Epub ahead of print,
doi: 10.1104/pp.18.01215.
www.plantphysiol.org/content/early/2019/01/28/pp.18.01215.long
Contact:
Dr. Nico Dissmeyer
Phone: 0345 5582 1710
nico.dissmeyer@ipb-halle.de