Background and Significance
Introduction
Panama is a significant banana producer in Latin America, yielding 2.5 million tons annually, contributing about $200 million to Panama's economy. Currently, there are no ultimate solutions to manage the spread of Fusarium oxysporum. This asexual-reproducing fungal pathogen can remain in an endophyte form in soil, tissue, and organic residues asymptomatically.
The banana industry in Panama has remained an important source of employment and economic development in local communities. It is also a significant part of the basic diet of the population. The most commonly cultivated banana is the species Musa x paradisiaca, also known as the Cavendish. Moreover, the industry makes approximately 200 million dollars (Ministerio de Desarrollo Agropecuario, 2023).
Global Impact of Banana Production
400 million people worldwide depend on bananas as a primary part of their diet. In Latin America alone, 300,000 people are employed to work plantations to provide bananas for the entire continent. The most prominent player in the industry is Chiquita, previously the United Fruit Company, which has been involved in the area since the turn of the 19th century.
Historical Context: Banana Production in Panama
Banana production in Panama has suffered a steady decline since 1985 when its export contributed $78 million to the local economy. At the time, the Chiriqui Land Company, a subsidiary of the United Fruit Company, owned 70% of the banana production in the country. In regions like Bocas del Toro, the company built the initial infrastructure. As they exploited the region for bananas they essentially acted as a parallel government, as the land was theirs and not of the state. Through geopolitics, 15,700 hectares of land were given back, but Chiquita can still dictate whether infrastructure is suitable to repurpose.
The Emergence of Fusarium Wilt and Its Impact
One of the reasons that Chiquita could hold so much power over the region was the emergence of Fusarium wilt. Local farmers did not have the knowledge or resources to combat a fungus that was depleting their harvest of an important crop. Therefore, in the 1950s governments all around Latin America chose to give Chiquita complete jurisdiction over this land.
Chiquita parted ways with Panama in 2008, where the contribution of bananas to the country's GDP fell to an alarming figure of just 33 million dollars, as the production of bananas was under the 2 million mark. Since then, Panamanians attempted to nationalize banana production through disorganized and uninformed efforts which led to Chiquita’s return to the region. This didn’t succeed due to trade disagreements, labor unions putting disputes forward, and unorganized attempts at creating new enterprises.
The Problem: Panama Wilt Disease
Our project addresses the issue of the Panama wilt disease caused by Fusarium oxysporum Spp. cubense (Foc), which clogs the plant's xylem vessels; leading to water and nutrient deprivation, defoliation, and plant death. The problem was initially mediated by changing the species of Banana cultivars from Gross Michael to Cavendish, but Tropical Race 4 has rendered this species vulnerable as well.
Current Challenges in Disease Management
The current strategies for disease remediation are isolation and quarantine which are ineffective in treating the infection and are usually conducted at a late stage of infection when symptoms are already visible and indicate a substantial amount of harm to the banana plantation. Managing this pathogen is challenging because it can persist in soil or host tissue, grow in organic residues, and survive as asymptomatic endophytes in non-host plants. The disease can have a long incubation period, and infected plants cannot be detected until external symptoms appear.
Disease Progression and Symptoms
The time between root infection and the development of Fusarium wilt symptoms can vary from two to six months and is influenced by various factors. Initial symptoms consist of vein-clearing leaf epinasty, stunted growth, yellowing of lower leaves, intensifying wilting, defoliation, and the death of the plant. The fungal colonization causes the vascular tissue to turn brown, evident in the cross-sections of the stem.
Proposed Solution: Genetic Modification for Early Diagnosis
Our solution involves genetically modifying bacteria for early detection of F. oxysporum. A genetic circuit with a fusaric acid inducible promoter is activated by fusaric acid produced by the fungus. Upon detection, bacteria produce AHL through the Lux I gene to communicate via quorum sensing.
Synthetic biology provides a platform for creating novel biological solutions that can be finely tuned to specific needs, such as the detection and response to plant pathogens. This approach is particularly valuable in addressing challenges like the Panama disease, where traditional agricultural practices and chemical treatments have fallen short. By engineering biological systems, we can create more targeted and environmentally friendly interventions.
Quorum Sensing and Gene Expression
Quorum Sensing is dependent on the cell density of the bacteria so plasmids and bacterial strains have been chosen mindfully to ensure the proliferation of the extracellular AHL concentration. Without a heavy concentration of this AHL the signal won’t be strong enough.
Quorum sensing is a process of communication between bacteria in a colony and is managed through autoinducers, which are signaling molecules. This communication in quorum sensing is what allows bacteria to perform colony-wide tasks such as biofilm formation, virulence, and bioluminescence. Each bacteria in the colony synthesizes autoinducers, secreted out of the cell, during reproduction. While gram-positive bacteria produce peptide autoinducers which are actively transported through their cell wall, gram-negative bacteria release acyl-homoserine lactone (AHL) autoinducers which are able to diffuse passively through their cell wall. As the bacterial population continues to grow, the concentration of autoinducer molecules in the colony’s surrounding environment increases exponentially. Once the concentration reaches a threshold called the “critical mass”, the amount of extracellular autoinducers is no longer energetically favorable. This occurrence is due to the tendency of molecules to maintain a relative equilibrium of solute concentration by moving in a concentration gradient, from hypertonic to hypotonic areas. Once the autoinducers enter the cell, they bind to its receptors, activating signaling pathways and altering transcription factors. The altered transcription factors prompt the colony to perform the new functions, mentioned earlier (biofilm production, virulence, bioluminescence, etc). This shows that quorum sensing may only occur when the bacteria are in high enough concentrations. Quorum sensing in some bacteria may follow a negative feedback loop - they downregulate autoinducer production in order to prevent the bacterial colony from growing excessively.
Once the AHL forms the complex, with a Lux R gene coding for a regulatory protein on another plasmid, this becomes a transcription factor that helps activate the gene expression of Lux A and B which code for a heterodimeric Luciferase.
Bioluminescence Mechanism
A separate plasmid was modified with the AHL and a Lux R analog that triggers the Lux Operon, creating bioluminescence. FMN, and long fatty acids will react with Lux A and B luciferase to create light using the Vibrio Fischeri model. This will act as a reporter protein for our sensor, which will communicate that Fusarium Oxysporum is present in a given soil sample.
Both bioluminescence and fluorescence may appear similar on the surface: both of their light is produced when electrons move up the energy levels as they absorb energy, and when those ionized electrons return to lower energy levels their energy is released in the form of photons. However, key differences between their mechanisms of producing light affect the characteristics of their light and therefore how they are applied in synthetic biology. Firstly, the source of bioluminescence are the enzyme-substrate chemical reactions that occur within an organism. In our case, the luciferase enzyme acts on the luciferin substrate to create the luminosity. On the other hand, fluorescent light originates from molecules absorbing a high-energy wavelength and re-emitting the wavelength at a lower frequency. The process involved no chemical reactions. An advantage of employing bioluminescence instead of fluorescence is therefore that there is no need for external light in order to ionize the electrons. This not only allows the process to have a longer duration and occur in a manner that is more straightforward, but it also avoids having to use light that may cause issues such as phototoxicity, photobleaching, and autofluorescence from the specimen. Still, limitations of the bioluminescence mechanism include the limited potential applications of bacterial bioluminescence due to its low brightness levels. Low brightness bioluminescence is not effectively detected by cameras (particularly those used in projects like outs). This issue is especially prominent in biological systems that change rapidly as the cameras require longer exposure time to the bioluminescence.
Detection System: Hardware and Software Integration
To detect and quantify that bioluminescence create a hardware system that is capable of detecting the communication between the bacteria to subsequently alert farmers about the presence of the pathogenic fungus in the soil.
We initially planned to use an approach utilizing software and hardware through a Smartphone Detection System. A chamber and an algorithm are used to amplify the light detected through a smartphone camera. (Kim, H., Jung, Y., Doh, IJ. et al.) The algorithm takes up to 40 images and averages light at different wavelengths instead of increasing exposure like the typical method, reducing noise from the amplification. These averages are then put into a Simple Signal to Noise Ratio (SNR), which consists of (IPhotonsM)/ITotal , involving the current produced by incoming light, the amount of amplification, and the amount of noise present by the protons generated, dark current and the amplification itself.(Kim, H., Jung, Y., Doh, IJ. et al.)
However, due to time constraints and the complexity of the system, we were not able to implement this so we used a system involving a Raspberry Pi camera instead.
The Raspberry Pi camera is what will detect the bioluminescence emitted by the E. coli bacteria when in the presence of Fusarium Oxysporum. As the hardware is designed to be used in rural areas, as well as potentially by people of lower income, we focused on creating a system that is physically and also internally robust enough to endure the conditions of agricultural plantations for long periods of time. We also aimed to create a system that is intuitive to use. We started the development of a system using the Raspberry Pi camera by determining its light sensitivity through a test. In order to improve the signal-to-noise ratio of the system and potentially simulate the conditions of the banana plantations where our hardware is meant to be used, we created a sealed environment, free of external incoming light. This would allow our subsequently collected data to be clear and accurate. We wrapped a cardboard box with aluminum foil and sealed any areas with the potential to bring light from the outside environment into the box with black electrical tape. The Raspberry Pi camera was inserted into the box through a small opening, and a test tube through an opening at the top of the box (both parts were covered with black electrical tape). For the storage of the imaging conducted by the camera, we have inputted a Micro SD card into the cardboard. This will be controlled by our network connected to the Raspberry Pi, the person controlling it being able to take pictures of the inside of the box on command.
We solved the previously mentioned limitation of bacterial bioluminescence not being bright enough to be detected by cameras by editing the genetic sequence of the bacteria’s DNA. More specifically, we fused the genetic sequence of LuxB with a Venus Mutation called Bioluminescence Resonance Energy Transfer (BRET). This mutation has been justified to amplify the bioluminescence of the E. coli enough so as to be detected by our Raspberry Pi camera. Venus was fused either to the N- or C-terminus of LuxA or LuxB. When LuxB and Venus are in close proximity (typically within 10 nm), the LuxB acts as the donor, donating its bioluminescence to the fluorescence acceptor, which is the Venus. This dynamic is able to sustain itself because the emission spectra of LuxB, at 490 nm, overlap with Venus’ absorption spectra which is at 528 nm; this is the basis of BRET. Overall, the study demonstrated that the LuxVenus + Lux A mutation allowed for the greatest increase in bioluminescence for the bacteria, the bioluminescence measured to be 5 times greater than for LuxA + LuxB. Moreover, the mutation caused the hue of the bioluminescence to change from a blue-green color to a green color. This change is significant because the Raspberry Pi camera is less inclined to detect colors that are vague or are a mix of two tones.
Potential Impact and Conclusion
This project could be a major step forward in solving the threat the Panama disease poses to the global food supply and the 400 million people who depend on bananas as a key ingredient for their food. Not only could an early detection of F. oxysporum help farmers save millions of bananas, but their revenue can contribute to the 13.5 billion dollar Banana industry.
Implementing this project has the potential to significantly alter the management of Panama disease by shifting the focus from reaction to prevention. Early detection allows for more precise interventions, potentially saving large swathes of banana crops from devastation. Moreover, this approach can reduce the reliance on chemical treatments, aligning with global efforts towards sustainable agriculture.
In conclusion, by integrating synthetic biology with practical agricultural needs, our iGEM project tackles a pressing global issue with innovative technology. The choice to focus on the Panama disease reflects both an urgent need and a significant opportunity to impact food security worldwide. Through this project, we not only aim to address a specific agricultural problem but also to contribute to the broader application of synthetic biology in solving real-world challenges.
References
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Kang Y., Cheng Z., Glick B. R., & Guo J. (2023). Quorum sensing and its applications in agriculture. International Journal of Molecular Sciences, 24(6), 5096. https://doi.org/10.3390/ijms24065096
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