Overview
CAP'siRNA is an innovative RNA platform that uses long interfering RNA precursors encapsulated in viral capsids to eliminate the Beet Yellows Virus (BYV) in sugar beets. We offer a solution that is efficient, specific, non-GMO, biodegradable, and capable of reducing the sugar beets loss, hence increasing sugar production. Ultimately, this project could be declined to address various types of agricultural diseases.
Introduction
Global hunger has always been a real concern throughout history. According to the Food and Agriculture Organization of the United Nations (FAO), 733.4 millions of people in the world are undernourished [1]. More and more resources are drained from the earth, leading to global scarcity. To tackle this issue, particular focus has been placed on agriculture and finding new ways to increase food production. In recent years, food production has significantly increased, but it struggles to keep pace with overpopulation, climate change, and emerging diseases. A tremendous number of diseases exist for plants, being of bacterial, fungal or even viral origin. As plants do not have an immune system like humans or many animals do, they had to evolve to protect themselves [2]. They developed in that case resistance genes. These genes protect the plants against pathogens, but also harsh conditions [3].
Sugar is one of the most widely produced and exported goods worldwide, with 178.930 millions of tons produced in 2022 [4]. 25% of its production comes from sugar beets, and the European leader and second-largest global producer is France [5]. The majority of this sugar is used in the agri-food business, but 33% is not destined for direct consumption. Instead, it is transformed into alcohol, which is utilized in various products, ranging from hand sanitizer, cosmetics to even biofuel for powering cars [6].
Problems
These sugar beets are planted and collected at a specific time: from the end of March to the beginning of November [7]. Unfortunately, during this same period, specific aphids called Myzus Persicae emerge from hibernation, carrying various viruses. These include the four viruses responsible for Virus Yellows disease which are Beet Yellows Virus (BYV), Beet Mild Yellowing Virus (BMYV), Beet Chlorosis Virus (BChV), and Beet Mosaic Virus (BtMV). This disease causes the yellowing of leaves, leading to brittleness, necrosis and reduced roots mass. This leads to the death of sugar beets, and when it doesn't kill the plant, weakens it significantly and reduces its sugar yield. On average in France, Virus Yellows disease decreases sugar production by 30 to 40% and it can go up to 90% in the most affected areas. The most severe form of Virus Yellows disease is inflicted by the BYV, and its prevalence has been seen to increase due to climate change [8].
Figure 1: The transformation process of sugar beets into sugar
Aphids, particularly Myzus persicae and Aphis fabae, are harmful insects that play a major role in transmitting viruses, like those responsible for the beet yellows disease. Myzus persicae, or the green peach aphid, is especially dangerous due to its ability to transmit several plant viruses to different crops, including sugar beets, tomatoes and potatoes [8]. This aphid is widespread across the world, particularly in Europe and Asia, and it easily adapts to various climates. Its life cycle is rapid, with successive generations multiplying quickly, especially in the summer through asexual reproduction. When aphid populations reach a certain density, winged forms appear, allowing them to spread to new host plants. These winged forms are crucial for the large-scale spread of infestations. Additionally, aphids have become increasingly resistant to many insecticides, making their control even more challenging and requiring innovative management strategies to protect agricultural crops [10].
Current solutions
Different solutions exist to counter the problem by targeting the aphids. The most famous one is the neonicotinoids, but this insecticide was banned in 2023 in France due to its harmful effects on pollinators and human health. In Europe, it is only authorized under specific guidelines [11, 12]. Other solutions include co-culture, which can attract aphids to other plants. Moreover, various chemical pheromones such as allomones and kairomones can be sprayed to control aphid location. The use of predators like ladybugs or nematodes can also be introduced to target the insects [13, 14]. Unfortunately, these solutions are not efficient enough, are expensive on a large scale, and kill aphids. Killing them risks reducing biodiversity, as aphids are part of trophic chains that impact other organisms.
What are pheromones? The online library Britanica says “any endogenous chemical secreted in minute amounts by an organism in order to elicit a particular reaction from another organism of the same species” [15]. There exist two types of natural odors to control pests: kairomones and allomones. Kairomones are smells that attract pests, such as reproductive molecules of a specific species. Therefore, when the insects sense these molecules, they go towards their direction. On the contrary, allomones are repulsive smells that pushes away pests from their habitat. It is a natural defense mechanism used by plants for a long time. Nowadays, these pheromones are produced as defensive means in agriculture to control specific pests' population [14].
Our innovation
This year, students from Ionis-Paris decided to tackle this problem using interfering RNA (RNAi) technology. The idea is to engineer bacteria Escherichia Coli, with long precursors of short interfering RNAs (siRNA), that will target and destroy specifically the viral RNA genome of the BYV. However, RNA is very unstable and easily degradable in the outside environment by enzymatic or chemical reactions. Therefore, we decided to combine this solution with a second one, hijacking the properties of viral capsids and using them as vectors for the RNA delivery into the plant. Our RNA complex will be protected by the viral capsid and delivered once in contact within the extracellular matrix of the plant cells. Inside the plant, the RNA will spontaneously spread out everywhere, and associate with the plant's enzymatic complexes to degrade the RNA genome of the BYV.
RNA interference
RNA interference, or RNAi, is a known defense mechanism in plants that recognizes exogenously invading genetic material such as viruses. When a virus enters the plant, its mRNA sequence is recognized and the plant itself produces mRNA strands that will be degraded using the RNA interference pathway. We decided to implement the same strategy, by designing double stranded RNA strands that will be administered into the plant and will follow the same degradation pathway. They will firstly be taken up by an enzymatic complex called Dicer. This complex will bind to the RNA strand, select a length of approximately 21-23 nucleotides, and thanks to its endonucleases activity, will cleave the RNA strand. This will create short interfering RNAs or siRNAs of around 21 nucleotides. These siRNAs will then be incorporated into another enzymatic complex called RNA-induced silencing complex or RISC. The siRNA will act as a guide strand to the RISC, leading the complex to the complementary sequence of the siRNA localized on the RNA viral genome. Once bound to the viral mRNA, the Argonaute-2-endonuclease or Ago2, a protein within the RISC, will cleave the target mRNA at a specific site, inhibiting viral protein expression [16].
Figure 2: RNA interference mechanism
Long hairpin RNAi precursors
For an increased stability, we synthesize long hairpin RNA (lhRNA) strands, that are double stranded RNA shaped in a hairpin-like form. Long RNAi precursors enable us to create numerous siRNA from one long hairpin RNA. This will give us many advantages: we would have more RNA in the plants, making the solution more lasting and efficient, and is supposed to be more efficient than simply shRNA in plants [17]. RNA viruses are known to mutate very fast, therefore as one siRNA is enough to eliminate one BYV, it would counter the problem of viral mutation [18]. To know more about the system countering the mutations of the BYV, see in PrediRNA part. However, synthetizing long hairpin RNAs enable us to adjust and put any sequence we want. Therefore, we can add multiple sequences that can target different genes in one RNA. It is all in one!
Viral capsid
Viruses have capsids to protect their genetic material from the surroundings environment. It is made of identical copies of coat proteins that bind between each other around a specific sequence on a viral genome. The use of empty viruses, therefore only the capsid, has been used in medicine for many years to play the role of transporters and allow the delivery of specific components. But it has never been done in agriculture.
In order to use viruses as transporters for our lhRNA, the goal is to add a specific sequence called OAS (Origin of Assembly Sequence) to the sequence of our lhRNA. This sequence is tailor made for the Tobacco Mosaic Virus (TMV) RNA genome. This enables the viral coat proteins to bind to the OAS, and then they will bind to each other forming a protective barrier of the length of the RNAi precursors [19, 20]. This way, the RNAi precursors will be protected from any environmental constraints, and the viral capsid will open and release its RNA cargo as soon as it enters the plant.
The disassembly of the viral capsid is a critical step in the viral life cycle, enabling the release of the viral genome into the host cell for replication. This process is regulated by environmental changes such as pH and ion concentrations. In the presence of calcium ions (Ca2+) at acidic pH, specific interactions within the capsid, including Ca2+ coordination and bonding between key residues glutamate-95 (E95) and glutamate-97 (E97), stabilize the capsid structure. Upon entering the host cell, the pH increases, causing the deprotonation of the carboxyl groups on E95 and E97, which leads to repulsive forces that destabilize these interactions. This causes E97 to move away from E95, triggering further conformational changes involving residues asparagine-98 (N98) and asparagine-101 (N101), which destabilize the Ca2+ binding site and promote Ca2+ removal. These concerted changes weaken the inter-subunit connections within the capsid, initiating a cooperative disassembly process that starts at the less stable ends of the virion. This controlled disassembly ensures the capsid releases its genetic material at the appropriate time and place, facilitating the virus’ replication within the host cell [21].
Figure 3: Explanatory video on the encapsidation of lhRNAs up to their release in sugar beet leaves
Application of our solution
Studies show that plants can accept exogenous interfering RNA precursors when they are sprayed onto their leaves [22]. Moreover, sprayed wild type TMV can induce death to the receiving plant, suggesting the entry of the virus in the said plant [23]. So we hypothesized that if the TMV virus enters the plant, the TMV capsid must also enter it. Therefore, combining those two ideas, our solution could be effectively propagated and processed into plants. Overall, viral capsid surrounds the RNAi precursors by recognizing the OAS, and forms a protective barrier against the environment, stabilizing the RNAi and permitting their release once they enter the plant. The product will be given to farmers as a spraying solution. They will directly apply it using their traditional spraying equipment. Following the aphids' season, which generally lasts two months from March to May, we advise them to spread it twice a year. They will spread the solution firstly in the beginning of March just before the aphids' arrival, and a month later in April. The solution is supposed to be both preventive, as it can be stored for a certain time in the plant before arrival of the BYV, and curative, as it can stop the spreading of the BYV that is already present in the plant.
Future prospect
In developing our system, we were limited by the implementation of the solution, as EU regulations forbid us to deploy GMOs in the environment. Therefore, we analyzed few improvements that our solution could have and explored paths that could lead to future work: In our project, we directly address the threat of the deadliest virus causing the Yellows disease, the BYV. However, with our technology, we could also introduce sequences against the 4 viruses involved for the Yellows disease (BYV, BMYV, BChV and BtMV). The sequences against the 4 viruses could be reunited into one strand and eliminate the yellows disease totally. Laboratory test are needed for that as we have already created the plasmid in silico.
Figure 3: Plasmid map of lhRNA against the 4 viruses involved in the Yellows Virus disease
Moreover, our system could be really curative. Indeed, in our lhRNAs, we could add a sequence that improves the synthesis of chlorophyll, or is able to reverse the symptoms of the yellows. This approach would eliminate the virus and help restore the green color to the sugar beet leaves, allowing for larger roots and an increased sugar yield. To finish with the sequences, we could introduce any desired RNA strand to combat various viruses currently affecting agriculture nowadays. This system could be replicated to anything we have in mind, and that’s the strength of CAP’siRNA!
As entering the plant can be complicated, we considered modifying the capsid proteins. Specifically, we propose adding a Cell Penetrating Peptide (CPP) to the coat proteins. This modification would facilitate the penetration of the capsid, which contains the RNAis, through the cell membrane. Consequently, the capsid would be able to enter the cell from the outside environment more easily. This would allow our solution to penetrate the cuticle faster and more efficiently. Unfortunately, this would be considered a GMO, thus not be applicable in Europe.
References
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