Part:BBa_K3610043
EFR ectodomain / SmallBit for S. cerevisiae
This part includes the ectodomain of the plant pattern recognition receptor EFR fused to the SmallBit part of the split-NanoLuc system. To ensure localization at the membrane, this part further contains the sequence for the signal peptide of the alpha factor from S. cerevisiae.
Usage and Biology
EFR
Elongation factor-thermo unstable receptor (EFR) from A. thaliana is a plant pattern-recognition receptor (PRR). It is a cell surface receptor and part of the plants firts defence mechanism against potential pathogens. The EFR receptor is also a leucine-rich-repeats (LRR) receptor-like serine/threonine-protein kinase. The protein consists of an extracellular domain with leucine-rich repeats, a ligand binding domain found in many receptors, a single-pass transmembrane domain and finally an intracellular kinase domain. The ligand binding domain from EFR has high specificity to a bacterial pathogen-associated moleculat pattern (PAMP), namely the epitope elf18 of the abundant protein Elongation Factor Tu (EF-Tu), which is catalyzes the binding of aminoacyl-tRNA (aa-tRNA) to the ribosome in most prokaryotes and therefore is evolutionarily highly conserved. This makes the EFR a receptor that can be activated by the presence of a huge variety of bacteria. Upon binding of the ligand to the extracellular domain, the receptor dimerizes with its coreceptor BRI1-associated receptor kinase (BAK1). This interaction triggers the activation of the intracellular kinase domain of EFR and BAK1, initiating a signal cascade leading to an upregulation of immune response mechanisms.
Usage with NanoLuc
In this case, the C-terminal domain of EFR, entailing the intracellular kinase domain, was removed from the sequence. Instead, the SmallBit part of the split NanoLuc luciferase was fused to the C-terminal domain via a 15 amino acid linker.
The ligand-dependent interaction of EFR with its coreceptor BAK1 is driven by the extracellular ligand-binding domain. Further necessary is the transmembrane domain, including the juxtamembrane domain. Therefore, dimerization can be achieved without the intracellular kinase domain of neither EFR nor BAK1. Coexpressed with the ectodomain of BAK1 fused to the LargeBit part of the NanoLuc luciferase, elf18-induced interaction between BAK1 and EFR can drive the reassembly of both parts from the NanoLuc luciferase, reconstituting its function to react with furimazine in the presence of oxigen, yielding furimamide and a fluorescent output. This part, therefore, allows visualization of the ligand-dependent interaction of the plant PRRs EFR and BAK1. This enables us to use this part, in coordination with the BAK1 ectodomain and LargeBit NanoLuc, to visually capture the presence of the elf18 epitope in water samples as the elf18 pattern will induce interaction between the receptors, causing the split-NanoLuc luciferase parts to rejoin and generate a functional protein, which gives a visual uotput with the substrate furimazine.
Characterization
We coexpressed this part together with Part:BBa_K3610038 (eBAK1), which is the ectodomain of the co-receptor BAK1 fused to the LargeBit part of the NanoBit system, in S. cerevisiae. The parts were assembled with Golden Gate Cloning in two different vectors. This part (eEFR) was assembled in a plasmid containing a kanamycin resistance gene, while the eBAK1 construct was assembled in a plasmid with a TRP1 gene, which encodes an enzyme necessary for tryptophan synthesis. These two genes allowed for selction on selective media.
After coexpressing the two plasmids in S. cerevisiae, a dimerization assay under a luminometer was performed with a plate reader of the type Synergy H1.
we tested whether the split-NanoLuc proteins would be able to interact, which would reconstitute its functionality. We further were interested in the interaction between the EFR and BAK1 ectoomain. EFR and BAK1 naturally interact upon binding of the epitope elf18. Should both receptors be expressed properly in the cell membrane of S. cerevisiae, the ligand will be able to bind to the EFR ectodomain. If this is sufficient to drive dimerization of the two receptor in S. cerevisiae, then the NanoLuc parts will be more likely to interact as well, which would lead to a higher amount of functional luciferase proteins in the cell, which would enable it to catalyze the reaction of furimazine to furimamide, a reaction which is accompanied by luminescence.
Luminescence assay
Samples with cells which were transfected with the two mentioned plasmids (eBAK1 and eCORE) and samples containing S. cerevisiae cells that were not transfected with any plasmids (UT).
Optical densities (OD600) of all samples were adjusted to 0.34.
For each type of sample, three types of measurements were made:
- 1 µL of deionized water added (no elicitor)
- 1 µL of epitope elf18 added
- 1 µL of epitope csp22 added
Each measurement was done four times with sample size 50µL. To each well 50 µL NanoGlo solution was added (50:1 buffer to furimazine)
The average of each type of measurement is displayed in the figure below.
As expected, the untransfected samples were not giving a luminescent output. Cells containing the plasmids with the two ectodomains displayed a strong increase in luminescence. However, the highest levels were measured when no bacterial epitope was added.
The results suggest that the plasmids had been taken up and get expressed in S. cerevisiae. It has also been shown that the split-proteins, which were fused to the receptors were able to interact and regain functionality as an enzyme. It seems, however, that this reconstitution is not driven by ligand-dependent interaction of the receptors. It is possible that the receptors are able to interact but lack any regulatory features which are otherwise present in plants, such as the BIR receptors. Another reason for these results could be the lack of glycosylation. In plants the receptors get heavily glycosylated. It is at this point unclear how exactly the receptors are glycosylated in S. cerevisiae and how this influences the functionality of the receptors.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 355
Illegal NheI site found at 1285
Illegal NheI site found at 2203 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1871
- 1000COMPATIBLE WITH RFC[1000]
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