Team:TU-Munich/Project/Killswitch

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A novel mechanism preventing uncontrolled spread of transgenic plants

Background – Why generate a plant which kills itself under certain conditions?

Working on plants is uncomplicated since as photoautotrophic organisms they can provide their own energy. So creating a photosensitive plant might seem silly at first glance. "Crazy, stupid Germans!", you might think, but wait, there's more! Green biotechnology doesnt have an easy stand in Germany since the "German Angst" of uncontrolled spreading of transgenic plants. Therefore, we see it as our task and duty to meet the required high safety standards that minimize these risks for a maximum of biosafety. We created a plant that can only survive in a well defined environment. Plants do not neccessarily need the whole spectrum of light to supply themselves with energy, so reassigning part of the spectrum to other purposes is possible. Shielded from red light by filters, the moss survives without compromising vitality or growth. Unintended release of our protected environment leads to activation of a lethal process of no return and thus kills the moss.

Design of a red light triggered nuclease system for self-destruction

Phytochrome B (abbrev.: PhyB) is a plant photoreceptor that exists in two interconvertible forms P_r and P_fr. In its P_fr (far red light sensititve) form upon red light illumination, PhyB interacts with Phytochrome Interacting Factors, e. g. PIF3 or PIF6.

Phytochrome Interacting Factor 3/6 (abbrev.: PIF3/PIF6) are both transcription factors with basic helix-loop-helix (bHLH) motifs that that bind to PhyB upon red light exposition

TEV Protease is a site-specific cystein protease from the C4 peptidase family from Tabacco Etch Virus (TEV). Its typical recognition site is ENLYFQ(G/S).

Split TEV Protease is a split version of the TEV Protease which N- and C-terminal split parts shows no enzymatic activity. Bringing them to physical proximity leads to reconstitution of the cleavage activity.

Micococcal nuclease (also called: Thermonuclease, MNase) is a non-specific endo-exonuclease from Staphylococcus aureus which shows activy in digesting single-stranded DNA/RNA and also double-stranded DNA/RNA.

In plants, the complete regulation and mechanism of programmed cell-death (abbrev.: PCD) is still not fully understood, but it is known that in the most eukaryotic organisms fragmentation of the genome inevitably leads to PCD.

Micrococcal nuclease is choosen as the effector to destroy the integrity of the genomic DNA. But how we can control the spatiotemporal death-bringing activity of the nuclease which only gets activated unter certain conditions?

Our solution is to anchor the nuclease in the cytoplasmatic membrane under basal conditions. In its membrane-associated form, the nuclease can not evolve its deadly potential. The reason is that the genomic DNA of eukaryotic organisms is subcellular located in the nucleus, sheltered by the nuclear envelope.

The question is how to enable the lethal activity of the nuclease upon red light illumination? The first component consists a fusion protein composed of a signal peptide plus transmembrane region for membrane localization, a TEV cleavage site for cleaving off the nuclease upon red light exposition and nuclear localizaton signal plus the nuclease itself as the death bringing part.

Red light triggered reconstitution of split TEV Protease

To liberate the nuclease after exposition to red light we designed two fusion proteins; each of them contains either the N-terminal or the C-terminal part of the TEV Protease. The first fusion protein has PhyB as its fusion partner which heterodimerizes with PIF3/PIF6 that is the fusion partner of the second fusion protein. Red light induces heterodimerization of PhyB with PIF3/PIF6 and thus reconstituting the N- and C-terminal half of the TEV Protease resulting in a proteolytic activity for a specific TEV recocnition site.

The reconstituted TEV Protease libarates the nuclease from its membrane anchore by cleaving it off from the transmembrane domain. The nuclear localization signal ensures nuclear translocation of the nuclease.

Reaching the nucleus, the nuclease can unfold its full deadly impact by digest the genomic DNA in fragments through its endo-/exonuclease activity. Genomic fragmentation automatically triggeres endogenous programmed cell-death.

PhyB-PIF3/PIF6 interaction under red light exposition

Reconstitution of Split-TEV-Protease

Liberation of membrane-anchored nuclease and nuclear translocation

Disruption of the genetic material and programmed cell-death

Design of our construct

Results

Text

figure

References:

http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984

http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984 Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. Cell, 37(2):629–33.

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 === Background &ndash; Why generate a plant which kills itself under certain conditions? ===
-Working on plants is uncomplicated since as photoautotrophic organisms they can provide their own energy. So creating a photosensitive plant might seem silly at first glance. "Crazy, stupid Germans!", you might think, but wait, there's  more! Green biotechnology doesnt have an easy stand in Germany since the "German Angst" of uncontrolled spreading of transgenic plants. Therefore, we see it as our task and duty to meet the required high safety standards that minimize these risks for a maximum of biosafety. We created a plant that can only survive in a well defined environment. Plants don't neccessarily need the whole spectrum of light to supply themselves with energy, so reassigning part of the spectrum to other purposes is possible. Shielded from red light by filters, the moss survives without compromising vitality or growth. Unintended release of our protected environment leads to activation of a lethal process of no return and thus kills the moss.
+Working on plants is uncomplicated since as photoautotrophic organisms they can provide their own energy. So creating a photosensitive plant might seem silly at first glance. "Crazy, stupid Germans!", you might think, but wait, there's  more! Green biotechnology doesnt have an easy stand in Germany since the "German Angst" of uncontrolled spreading of transgenic plants. Therefore, we see it as our task and duty to meet the required high safety standards that minimize these risks for a maximum of biosafety. We created a plant that can only survive in a well defined environment. Plants do not neccessarily need the whole spectrum of light to supply themselves with energy, so reassigning part of the spectrum to other purposes is possible. Shielded from red light by filters, the moss survives without compromising vitality or growth. Unintended release of our protected environment leads to activation of a lethal process of no return and thus kills the moss.
 === Design of a red light triggered nuclease system for self-destruction ===
@@ Line 26: / Line 26: @@
 '''Micococcal nuclease''' (also called: '''Thermonuclease, MNase''') is a non-specific endo-exonuclease from ''Staphylococcus aureus'' which shows activy in digesting single-stranded DNA/RNA and also double-stranded DNA/RNA.</div>
-In plants the complete regulation and mechanism of '''programmed cell-death''' (abbrev.: '''PCD''') is still not fully understood. But it is known that like in the most eukaryotic organisms fragmentation of the genome inevitably leads to '''PCD'''. '''Micrococcal nuclease''' is choosen as the effector to digest the genomic DNA. But how to produce a death-bringing protein which only gets activated unter certain conditions? The solution is to anchor the nuclease in the cytoplasmatic membrane. In its membrane associated form the nuclease can not evolve its deadly potential because in eukaryotic organisms the genomic DNA is subcellular located in the nucleus sheltered by the nuclear envelope. The question is how to enable the lethal activity of the nuclease upon red light illumination? The first component consists a fusion protein composed of a '''signal peptide plus transmembrane region''' for '''membrane localization''', a '''TEV cleavage site''' for cleaving off the nuclease upon red light exposition and nuclear localizaton signal plus the nuclease itself as the death bringing part.
+In plants, the complete regulation and mechanism of '''programmed cell-death''' (abbrev.: '''PCD''') is still not fully understood, but it is known that in the most eukaryotic organisms fragmentation of the genome inevitably leads to '''PCD'''.
+'''Micrococcal nuclease''' is choosen as the effector to destroy the integrity of the genomic DNA. But how we can control the spatiotemporal death-bringing activity of the nuclease which only gets activated unter certain conditions?
+Our solution is to anchor the nuclease in the cytoplasmatic membrane under basal conditions. In its membrane-associated form, the nuclease can not evolve its deadly potential. The reason is that the genomic DNA of eukaryotic organisms is subcellular located in the nucleus, sheltered by the nuclear envelope.
+The question is how to enable the lethal activity of the nuclease upon red light illumination? The first component consists a fusion protein composed of a '''signal peptide plus transmembrane region''' for '''membrane localization''', a '''TEV cleavage site''' for cleaving off the nuclease upon red light exposition and nuclear localizaton signal plus the nuclease itself as the death bringing part.
 === Red light triggered reconstitution of split TEV Protease ===