Team:UZurich/Design
Design
1. Engineered OMVs that trigger stronger plant immune response
From previous works we already know that bacterial outer membrane vesicles (OMVs) can elicit protective plant immune response [1][2]. In order to enhance the immune response triggered by natural OMVs, we planned to display some immunogenic epitopes on the OMV surface. For this purpose, we used ClyA, a well-studied outer membrane-associated protein which is particularly enriched in OMVs, to which we linked our elicitors flg22 (BBa_K3989010), elf18 (BBa_K3989011) or both (BBa_K3989013). Therefore, as ClyA gets integrated into the membrane, our epitopes are displayed on cell surface and eventually get secreted within our OMVs.
For plasmid construction, we used a modular Golden Gate system [3] so that all parts are interchangeable, especially elicitor to be displayed, and outermembrane protein used for surface display. It is made up of the following basic parts:
- Promoter and RBS, in our case, arabinose inducible promotor (BBa_I0500) with medium RBS (BBa_B0032)
- Outer membrane-associated protein with linker for displaying, in our case, ClyA (BBa_K811000)
- Immunogenic epitopes or cellulose binding domain, in our case, flg22 (BBa_K3286138), elf18 (BBa_K3989023), and double cellulose binding domain (BBa_K1321340)
- Terminator, in our case, double terminator (BBa_B0015)
Figure 1: ClyA construct with different possible epitopes
To achieve high immunogenicity, we want as much as ClyA-elicitor fusion protein to reach the outer membrane. For this reason, we chose the E. coli BL21 omp8 strain. In this strain, three main outer membrane proteins, OmpA, OmpC, and OmpF were deleted, leaving more space on the outer membrane for heterogeneous proteins expression (Figure 2) [4][5].
Figure 2: E. coli BL21 omp8 strain has more outer membrane space for heterogeneous protein [4]
For a more detailed description of our design and our design process, please visit our Engineering Success page.
2. Inducible OMV Production System
One way to apply our idea in the real world is to put an engineered bacteria strain containing an inducible OMV production system. The system is designed to make our bacteria produce OMVs only when specific pathogens are sensed.
It is made up of two parts. The first one is an inducible lipoprotein TolB system. This lipoprotein is responsible for outer membrane stability. Previous studies showed that the TolB deletion strain has a higher OMV production ability [6]. The second one is the TEV protease-mediated TolB degradation system. We added three TEV protease cutting sites to the original TolB protein so that when the TEV protease exists, the TolB protein can be cut and lose its function.
Figure 3a: AHL-sketch 1
Figure 3b: AHL-sketch 2
Figure 3c: AHL-sketch 3
Figure 3d: AHL-sketch 4
In our project, we put both of these two parts into a TolB deletion strain. Each part is controlled by a different promoter (shown in the figures above). The activation and repression of these two promoters are regulated by the quorum-sensing molecules (AHL). In the natural environment, the TolB encoding gene regulated by quorum sensing module EsaR-PesaR (BBa_K3989024) will be expressed constitutively. Thus, the OMV overproduction phenotype will be compensated and the strain will have normal behaviour. Meanwhile, since there are no AHL molecules, the LuxR promoter will not be activated and no TEV protease will be produced.
However, when AHL molecules exist (which means there are specific pathogens nearby), the expression of TolB will be suppressed and the TEV protease will be produced. The combination of these two events will dramatically decrease the amount of TolB protein in the bacteria. Thus, the periplasm will get destabilised and the bacteria will start to overproduce OMVs again.
3. CMV-based delivery system
Both designs mentioned above are about vesicles “touching” the plant, i.e. they both work at plant cell surface. However, is it possible to not only “touch” but also fuse the bacterial membrane vesicles with plant cells? This is a very important question that can improve our understanding not only on plant-microbe interactions but also on the design of novel biotechnological applications such as our OMVs.
Besides fundamental research, bacterial membrane vesicles also have high potential in delivering a wide range of molecular cargoes into plant cells, especially CMVs, since they contain all components of the bacterial cytoplasm. Thus, if fusion is possible, DNA, RNA, proteins and even small molecules can be delivered into plant cells.
- In the case of DNA delivery, the system could be adapted for transforming plant cells and modulating agronomic performance.[7]
- In the case of RNA delivery, it could be used for RNA interference experiments, which are a novel strategy for crop protection.[8]
- In the case of protein delivery, it could be employed to trigger another layer of plant immune response: effector-triggered immunity (ETI). Recent studies have shown that mutual potentiation of pattern-triggered immunity (PTI) and effector-triggered immunity can generate a more effective defense against pathogens.[9]
In order to test if CMV-plant-cell fusions are possible, we used our CMVs to deliver a viral vector containing a GFP gene to plants. If the CMVs can fuse to plant cells, we should be able to observe (through fluorescence microscopy) green fluorescence signals generated by the viral vector in the plant cells.
Important remarks
After introducing our three constructs it is important to mention the course of our project and how we planned to utilise these constructs in a feasible way. Due to the strict GEO laws is Switzerland we decided to focus more on an application that is feasible today and not only in the distant future. We would utilise the first construct to engineer OMVs that produce a stronger immune response and isolate them from the bacteria culture. These isolated OMVs would then be sprayed onto plants we want to treat before the season for a specific pathogen begins. Our second construct should demonstrate that a more sophisticated system could be possible in the future too. However, if it was to be implemented as suggested above, we would have to research ways more about ways to biologically contain the bacterium we would release into the soil for example kill switches, genetic parts that cannot work on other organisms, non-canonic AAs, etc. [10].
For more about the implementation of our project, visit our Implementation page.
[1] Bahar, O., Mordukhovich, G., Luu, D. D., Schwessinger, B., Daudi, A., Jehle, A. K., ... & Ronald, P. C. (2016). Bacterial outer membrane vesicles induce plant immune responses. Molecular Plant-Microbe Interactions, 29(5), 374-384.
[2] McMillan, H. M., Zebell, S. G., Ristaino, J. B., Dong, X., & Kuehn, M. J. (2021). Protective plant immune responses are elicited by bacterial outer membrane vesicles. Cell reports, 34(3), 108645.
[3] Chiasson, D., Giménez-Oya, V., Bircheneder, M., Bachmaier, S., Studtrucker, T., Ryan, J., ... & Parniske, M. (2019). A unified multi-kingdom Golden Gate cloning platform. Scientific reports, 9(1), 1-12.
[4] Thoma, J., Manioglu, S., Kalbermatter, D., Bosshart, P. D., Fotiadis, D., & Müller, D. J. (2018). Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies. Communications biology, 1(1), 1-9.
[5] Prilipov, A., Phale, P. S., Van Gelder, P., Rosenbusch, J. P., & Koebnik, R. (1998). Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli. FEMS microbiology letters, 163(1), 65-72.
[6] McBroom, A. J., Johnson, A. P., Vemulapalli, S., & Kuehn, M. J. (2006). Outer membrane vesicle production by Escherichia coli is independent of membrane instability. Journal of bacteriology, 188(15), 5385-5392.
[7] Torti, S., Schlesier, R., Thümmler, A., Bartels, D., Römer, P., Koch, B., ... & Gleba, Y. (2021). Transient reprogramming of crop plants for agronomic performance. Nature Plants, 7(2), 159-171.
[8] Niu, D., Hamby, R., Sanchez, J. N., Cai, Q., Yan, Q., & Jin, H. (2021). RNAs—a new frontier in crop protection. Current Opinion in Biotechnology, 70, 204-212.
[9] Ngou, B. P. M., Ahn, H. K., Ding, P., & Jones, J. D. (2021). Mutual potentiation of plant immunity by cell-surface and intracellular receptors. Nature, 592(7852), 110-115.
[10] Torres, L., Krüger, A., Csibra, E., Gianni, E., & Pinheiro, V. B. (2016). Synthetic biology approaches to biological containment: pre-emptively tackling potential risks. Essays in biochemistry, 60(4), 393–410. https://doi.org/10.1042/EBC20160013