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Project description

With climate change, temperatures throughout the world are becoming more extreme and unpredictable, adversely impacting entire ecosystems. At a smaller scale, farming is particularly affected by these drastic weather fluctuations. For instance, in the Swiss region of Valais, we have observed repetitive freezing spells in the past few springs, causing devastating losses in crops. Apricot trees are amongst the most affected, as a staggering 85% of fruits were lost this year. This dramatic outcome is not only a cultural loss for our region, as it endangers the celebrated “abricots du Valais” traditionally consumed each summer, but is also threatening the livelihoods of all those involved in the cultivation and processing of apricots. We therefore decided to dedicate our iGEM project to finding a way to protect plants from spring frost and to showing our support to the farmers that work tirelessly to feed us and our families.
To achieve this, we designed three complementary approaches that aim to tackle the various causes of plant freezing. We envisioned combining the products of each approach in a solution-based treatment that would be sprayed onto plants to safeguard them from frost damage.

Figure 1 | Schematics representing the three approaches of our project

Antifreeze Proteins

Our first approach set out to directly prevent the formation of ice crystals that are responsible for the deterioration of plant tissues. To do so, we used synthetic biology to microbially produce purified antifreeze proteins (AFPs), that are able to bind to ice crystals and, as a result, inhibit their growth. As different AFPs can have varying efficiencies in limiting crystal development, we chose to work with three candidates AFPs, namely Rhagium inquisitor AFP (RiAFP), Daucus carota AFP (DcAFP) and Flavobacterium frigoris PS1 AFP (FflBP). To produce these proteins, we built genetic constructs with which we transformed Escherichia coli bacteria. We then induced the expression of the desired protein in our engineered bacterium and followed an extraction protocol to obtain our different purified AFPs.

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The second approach we considered to protect the apricot flowers from freezing is based upon eliminating a plant pathogen, Pseudomonas syringae pv. syringae. This bacterium produces an ice nucleation protein (INP) that accelerates the formation of ice crystals on the plant. This results in increased frost damages at higher temperatures, making losses due to frost on apricot trees more extensive and frequent.
A way to kill this pathogen is by using tailocins. Tailocins are phage derived proteins produced by bacteria to fight their competitors – they have the structure of a phage tail and can perforate bacterial cell walls, killing the target cell. Because these proteins are very specific, they have the potential to eliminate the pathogenic strain P. syringae syringae, without harming the entire plant microbiota. Our goal was to first clone and express the tailocins in E. coli, allowing us to obtain purified tailocins to combine with our APFs in our solution to spray.

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The last approach we wished to implement to fight the detrimental effect of P. syringae syringae was to directly inactivate the InaZ gene responsible for the production of the INPs. We can do so by using a defective bacteriophage to inject a phagemid able to delete the InaZ sequence in the cells via CRISPR/Cas9 editing. The targeted deletion of the InaZ gene via CRISPR/Cas9 would allow the rest of the genome of P. syringae syringae to stay intact.
Using E. coli, we can produce the phage containing our phagemid in large quantities. The phages can then be purified and added to a spray to apply onto the plants. When infecting P. syringae syringae, the phage would introduce the phagemid into the cell, resulting in the deletion of the InaZ sequence and therefore preventing INP synthesis, effectively protecting the plant from further damage due to the pathogen.

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Testing our potential frost-protective spray

In parallel, we developed methods that could allow us to verify if our different approaches would work as intended. Due to time limitations, we especially tested our purified AFP and tailocin solutions.
To do so, we started by creating our very own cooling device, called FROZONE, that is able to precisely control the temperature of a copper plate onto which we can load sample solutions. In combination with a microscope, we used FROZONE to monitor and film the freezing process of our samples with or without our AFPs/tailocins. Thanks to FROZONE, we were able to observe, for instance, that while the presence of P. syringae syringae accelerates freezing of liquid as expected, the addition of our tailocins could interestingly reverse this phenomenon.
Then, we applied our AFPs on Arabidopsis leaves exposed to cold temperatures to test their efficacy in protecting plants from frost damages. We also developed our own algorithm, named VISION, to quantify the damages done to the plants, revealing that our AFP solutions successfully reduced tissue deterioration resulting from freezing.

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