Abstract
Toxic proteins are prime targets for molecular farming (the generation of pharmacologically active or biotechnologically usable compounds in plants) and are also efficient tools for targeted cell ablation in genetics, developmental biology, and biotechnology. However, achieving conditional activity of cytotoxins and maintaining the toxin-expressing plants as stably transformed lines remain challenging. Here, we produce a switchable version of the highly cytotoxic bacterial RNase barnase by fusing the protein to a portable protein degradation cassette, the low-temperature degron cassette. This method allows conditional genetics based on conditional protein degradation via the N-end rule or N-degron pathway and has been used to vice versa accumulate and/or deplete a diverse variety of highly active, unstable or stable target proteins in different living multicellular organisms and cell systems. Moreover, we expressed the barnase fusion under control of the trichome-specific TRIPTYCHON promoter. This enabled efficient temperature-dependent control of protein accumulation in Arabidopsis (Arabidopsis thaliana) leaf hairs (trichomes). By tuning the levels of the protein, we were able to control the fate of trichomes in vivo. The on-demand formation of trichomes through manipulating the balance between stabilization and destabilization of barnase provides proof of concept for a robust and powerful tool for conditional switchable cell arrest. We present this tool as a potential strategy for the manufacture and accumulation of cytotoxic proteins and toxic high-value products in plants or for conditional genetic cell ablation.
Highlights
Toxic proteins are prime targets for molecular farming and are efficient tools for targeted cell ablation in genetics, developmental biology, and biotechnology
We used a permissive temperature of 14°C, which allows the protein of interest (POI) to remain stable, and a restrictive temperature of 28°C, which induces degradation of the POI
When expressed in trichomes of wild-type Arabidopsis plants under the control of the TRY promoter (ProTRY), lt-BAR resulted in efficient ablation of trichomes
Summary
Toxic proteins are prime targets for molecular farming (the generation of pharmacologically active or biotechnologically usable compounds in plants) and are efficient tools for targeted cell ablation in genetics, developmental biology, and biotechnology. We produce a switchable version of the highly cytotoxic bacterial RNase barnase by fusing the protein to a portable protein degradation cassette, the low-temperature degron cassette This method allows conditional genetics based on conditional protein degradation via the N-end rule or N-degron pathway and has been used to vice versa accumulate and/or deplete a diverse variety of highly active, unstable or stable target proteins in different living multicellular organisms and cell systems. The on-demand formation of trichomes through manipulating the balance between stabilization and destabilization of barnase provides proof of concept for a robust and powerful tool for conditional switchable cell arrest We present this tool as a potential strategy for the manufacture and accumulation of cytotoxic proteins and toxic high-value products in plants or for conditional genetic cell ablation. Biologically active lectin proteins from plants and animals could mainly be produced in Escherichia coli, albeit with moderate yields (Lam and Ng, 2011)
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