Team:UZurich/Description
Description
The Challenge
Over the course of the 20th century, the world population’s increase was “three times greater than in the entire previous history of humanity” [1]. To support this explosion in numbers, it has become crucial to minimise crop losses caused by pathogens. In order to do so, people have started to rely on pesticides. But in recent years, these products have come under heavy scrutiny.
Pesticides were also publicly discussed in Switzerland in June when we voted on not only one, but two popular initiatives about the use of such products. One demanded a "Switzerland free of synthetic pesticides" [2]. The other sought to increase the incentive to stop using pesticides by cutting direct payments to farms that used them [3]. Their common aim is clear: Reducing the use of synthetic pesticides.
Both initiatives were met with heavy criticism from the side of the farmers, many hanging up posters asking the population to vote against them as they would be too extreme.
However, both the post-vote analysis and our own interviews have shown that if possible, we in Switzerland would like to turn our backs on synthetic pesticides in favour of an agriculture that future generations can still profit from [4].
“If I didn’t have to, I wouldn't use them, that’s clear.”Rudy Studer, winegrower,
talking about synthetic pesticides [5]
For a more basic description of our project, please visit our Simple Description page.
The Goal
This discourse prompted us to take up the challenge of protecting crops in a sustainable fashion. Our basic idea is to let the plant protect itself using its immune system. We want to stimulate the plant’s natural defence mechanisms before it is exposed to pathogens so that when real threats come, the plant is already prepared. Our project makes use of the following three things: a fake pathogen, a delivery system and the plant’s natural defence mechanism.
The Fake Pathogen
Pathogen-associated molecular patterns (PAMPs) are molecules that are often highly conserved in groups of pathogens [6]. They include things like bacterial flagellin, elongation factors and fungal chitin [6]. When a plant senses PAMPs using pattern-recognition receptors, its immune system starts to work [7]. The use of basic ‘building blocks’ of other organisms to sense their presence is beneficial since they are constantly expressed on the pathogens and are not toxic, which means they can be safely used to trigger plant immune response.
The Delivery System
Outer Membrane Vesicles (OMVs) are buddings of the outer membrane of gram-negative bacteria and are often considered a result of stress response [8]. They are produced naturally to mediate virulence, communication, riddance of waste products and more [9]. OMVs are about 20-250nm in diameter and the contents are from the periplasm [10]. In recent years, they are being studied as drug delivery systems due to favourable properties such as stability, tolerability by humans and biodegradability [11][12].
The Plant's Defence Mechanism
Plants have natural defence mechanisms in place, one of which is pattern triggered immunity (PTI). In contrast to humans, plants do not have specialised immune cells but rather make use of chemicals and physical barriers [6]. At the heart of PTI lie the pattern-recognition receptors (PRRs). They are localised on the plasma membrane and most of them are receptor-like kinases (RLKs) or receptor-like proteins (RLPs). When a ligand binds to a PRR, one can observe several downstream responses that mediate plant immunity, such as the accumulation of Ca2+, reactive oxygen species (ROS) and phytohormones, as well as changes in the transcriptome of the plant [6].
BOOM V
In our project, we want to take a slightly different approach to pest control. Instead of killing or attacking the pathogen directly as most pesticides do, we aim to support the plant’s efforts to protect itself. Concretely, this means stimulation of PTI using PAMPs.
The reason we want to trigger PTI is, on the one hand, a practical one: Since they represent the first line of defense, they are mostly localized on the cell membrane [13]. This makes it easier for us to reach them than if they were cytosolic. On the other hand, the sheer variety of responses by the plant will make the development of resistance difficult.
The expression of the PAMPs on the outer membrane of our chassis allows us to load them on OMVs. These OMVs can then be applied to the plant where they trigger PTI. We chose the immunogenic molecules flagellin-22 (flg22) and the elongation factor Tu (elf18) to express on the OMVs, using a ClyA construct. They are well studied PAMPs that are recognised by the receptors FLAGELLIN-SENSING 2 (FLS2) and EF-Tu RECEPTOR (EFR) respectively. We chose these elicitors because they have been shown to induce a strong response in a wide variety of plants [14]. This would allow the application of our product for many plant species.
Another advantage of BOOM V is the modular nature of its design. Depending on the plant species, PTI could be induced more or less strongly by certain PAMPs. By replacing flg22 or elf18 with other elicitors, we can adjust our OMVs to maximise the response of the plant.
Naturally produced OMVs have a limitation, namely that the produced amount is insufficient for commercial use. However, it is known that there are proteins that connect the inner and outer membranes of gram-negative bacteria. The deletion of these proteins can cause hypervesiculation. Using deletion strains, we explored which bacterium had the best trade-off between growth rate and vesiculation. This allowed us to narrow down the most suited chassis for our constructs to two: E. coli tolB and E. coli BL21 omp8 strains.
What makes BOOM V good?
- Small probability of resistance
- Modularity allows wide application
- Biodegradability and compatibility with humans
- Derived from nature
Implementation
When it comes to the application of our OMVs, we thought of two possibilities:
Spray purified OMVs
This option would be in line with current regulations in Switzerland. It would mean that we purify our OMV samples so that no genetically engineered bacteria would be set free when the product is applied. Because OMVs can be degraded or washed off by the rain, the spray would have to be applied repeatedly.
Release engineered probiotics
To circumvent the above mentioned reapplication, we designed an inducible system that can be implemented into our bacteria. The induction depends on the QS molecules from the AHL class. They are often specific to certain bacteria, so we can adjust our promoter-binding protein to react to QS molecules from a specific pathogen. In our design, sensing of AHL molecules would result in the repression of tolB production and expression of a TEV protease. The protease will then cut the implemented tolB molecules at the TEV cutting site we inserted. As a result, our bacterium has a ∆tolB phenotype and due to decreased membrane stability, vesiculation is increased.
In this case, however, one would have to implement safety measures to ensure that the bacteria do not spread.
We followed both ways as they have their advantages and disadvantages. Our interviews with the farmers and the current legislative situation have shown that the spray option would be more appropriate. However, we believe that in a future where GEOs are more accepted in Switzerland, releasing the beneficial soil bacteria would be ideal.
Our Contribution to Sustainability
Now more than ever, it is important to think about how the things we use will impact the environment. This is why the sustainability of BOOM V was always on our minds. With our project, we address two of the 17 sustainable development goals of the UN and contribute to more sustainable agricultural practices. Though the perception seems to be that sustainability and GEOs are incompatible, this does not have to be the case: Synthetic biology can offer solutions for a more sustainable agriculture. With BOOM V, we aim to show just one of the myriad of applications that are promoting the realization of the sustainable development goals.
[1] Population Growth, 07. October 2021
[2] Initiative "For A Switzerland Without Synthetic Pesticides", 07. October 2021
[3] Drinking Water Initiative, 07. October 2021
[4] Post-vote Analysis, 07. October 2021
[5] Beetles, Borders and Bacteria: An Interview with Rudy Studer by iGEM UZH 2021, 07. October 2021
[6] Hou, Shuguo et al. “Damage-Associated Molecular Pattern-Triggered Immunity in Plants.” Frontiers in plant science vol. 10 646. 22 May. 2019, doi:10.3389/fpls.2019.00646
[7] Zipfel, Cyril, and Silke Robatzek. “Pathogen-associated molecular pattern-triggered immunity: veni, vidi...?.” Plant physiology vol. 154,2 (2010): 551-4. doi:10.1104/pp.110.161547
[8] Li, Ruizhen, and Liu Qiong. "Engineered Bacterial Outer Membrane Vesicles as Multifunctional Delivery Platforms." Frontiers in Materials vol. 7 202. 10. July. 2020, doi:10.3389/fmats.2020.00202
[9] Bahar, Ofir et al. “Bacterial Outer Membrane Vesicles Induce Plant Immune Responses.” Molecular plant-microbe interactions: MPMI vol. 29,5 (2016): 374-84. doi:10.1094/MPMI-12-15-0270-R
[10] Kulp, Adam, and Meta J Kuehn. “Biological functions and biogenesis of secreted bacterial outer membrane vesicles.” Annual review of microbiology vol. 64 (2010): 163-84. doi:10.1146/annurev.micro.091208.073413
[11] Bitto, Natalie J, and Maria Kaparakis-Liaskos. “The Therapeutic Benefit of Bacterial Membrane Vesicles.” International journal of molecular sciences vol. 18,6 1287. 16 Jun. 2017, doi:10.3390/ijms18061287
[12] Delbos, Valérie et al. “Impact of MenBvac, an outer membrane vesicle (OMV) vaccine, on the meningococcal carriage.” Vaccine vol. 31,40 (2013): 4416-20. doi:10.1016/j.vaccine.2013.06.080
[13] Couto, Daniel, and Cyril Zipfel. “Regulation of pattern recognition receptor signalling in plants.” Nature reviews. Immunology vol. 16,9 (2016): 537-52. doi:10.1038/nri.2016.77
[14] Furukawa, Takehito et al. “Two distinct EF-Tu epitopes induce immune responses in rice and Arabidopsis.” Molecular plant-microbe interactions: MPMI vol. 27,2 (2014): 113-24. doi:10.1094/MPMI-10-13-0304-R