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

Choosing the effector that triggers cell-death quickly and safely

Programmed Cell-Death (PCD) is a endogenous mechanism of the cell itself for a regulated suicide. It is normally carried out e. g. upon tissue differentiation or due to a infection with a pathogen to prevent its propagiation.

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.

Nuclear Localization Signal/Sequence (abbrev.: NLS) is a short amino acid sequence of a nuclear targeted protein that is recognized by nuclear transporters, the so-called importins, and then is actively transported in the nucleus. The opposite of NLS are NESs (Nuclear Export Signal/Sequence).

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 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159105 BBa_K1159105]) is choosen as the effector to destroy the integrity of the genomic DNA. Since the genome is protected by the nuclear envelope, we have to tag our nuclease with a NLS (Nuclear Localization Signal) ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159109 BBa_K1159109]). But how we can control the spatiotemporal death-bringing activity of the nuclease which only should gets activated unter certain conditions?

Preventing the nuclease to develop its deadly potential under well-defined conditions

Transmembrane Domain (abbrev.: TMD) is the region of a membrane protein which traverses the plasma membrane.

Our solution is to anchor the nuclease in the cytoplasmatic membrane under basal conditions. In its membrane-associated form ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159111 BBa_K1159111]), 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. By anchoring the nuclease in the cell membrane, the nuclease is spatially seperated from its site of action.

To make the nuclease releasable, we have design a long linker with a TEV recognition/cleavage site between the transmemmbrane domain (TMD) and NLS-tagged nuclease. In the presence of TEV protease activity, the nuclease is liberated from the membrane and translocates in the nucleus due to its NLS fusion. There, it can fulfill its task as the executor of PCD.

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

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.

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

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.

@@ Line 41: / Line 41: @@
 === Red light triggered reconstitution of split TEV Protease ===
+<div class="box-right">
+'''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.
+'''Phytochrome B''' (abbrev.: '''PhyB''') is a plant photoreceptor that exists in two interconvertible forms P<sub>r</sub> and P<sub>fr</sub>. In its P<sub>fr</sub> (far red light sensititve) form upon red light illumination, PhyB interacts with Phytochrome Interacting Factors, e.&nbsp;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
+</div>
 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.