Projet Description

Background

Our planet is currently experiencing a strong demographic growth, with all the responsibilities that this entails.

As the world struggles to meet current demands, this population surge will place even greater pressure on food production systems. In fact, by 2050, with the population expected to reach approximately 9.7 billion, a 70% increase in food production will be necessary to meet global nutritional needs (FAO, 2021).

This exponential rise in demand for agricultural products underscores the urgent need to address resource limitations and ensure food security and stability for the future. Given this immense challenge, it's crucial to focus on the key agricultural sectors that underpin global food security. Cereals, in particular, are a cornerstone of global agriculture, with over 700 million hectares of land dedicated to their cultivation worldwide. To put this in perspective, this area is roughly equivalent to the combined size of India and Argentina.

These crops are not only vital for direct human consumption, providing essential nutrients and serving as dietary staples across many cultures, but they also play a crucial role in livestock feeding. The extensive land use underscores their importance in sustaining both the global food supply and agricultural economies. As a result, any challenges affecting cereal production have far-reaching implications, making them a primary focus for agricultural research and innovation. ^[1-2-3]

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However, the increasing demand for these crops is pushing farmers to optimize yields while contending with challenges such as climate change, soil degradation, and plant diseases. To meet these growing needs, modern agriculture must adopt innovative technologies and sustainable practices to maximize yields while minimizing environmental impacts (Garnett et al., 2013).

Improving crop resistance to diseases and effectively managing pathogen vectors is therefore essential to ensuring global food security.

Plant viruses and their vectors

A. Viruses and their impact on crops

Plant viruses are microscopic pathogens that infect plantations, causing a range of detrimental symptoms that severely affect plant growth and yield, leading to substantial economic losses. Viral infections can result in leaf deformities, discoloration, stunted growth, and, in severe cases, plant death. These viruses are transmitted through vectors such as insects or contaminated agricultural tools, making their management a critical challenge. Excluding viroids, which have a circular RNA genome, five of the six major virus families include plant viruses, demonstrating that all plants around the world are sensitive to these pathogens. Plant viruses are classified into three types based on their interactions with vectors:

- Non-circulative (NC): The virus attaches to the insect's stylet during feeding and is released when the insect secretes saliva at a new feeding site. In this case, the virus does not pass through the insect’s system but remains attached to the mouthparts by specific interactions.

- Circulative, non-propagative: The virus moves through the insect's food canal, crosses the midgut and hindgut to reach the hemocoel (the insect's body fluid). The viral particles then cross the accessory salivary glands and are transmitted back to the plant via the saliva canal.

- Circulative, propagative: Similar to the previous type, the virus circulates within the insect but also infects and replicates within the insect’s cells. This mode of transmission allows for more persistent and effective spread of the virus among insect populations. As these viruses continue to pose a significant threat to agriculture, understanding their biology and modes of virus transmission is more crucial than ever to safeguard global food security. The table above consolidates information on the various families and a significant number of known plant viruses, detailing their vectors, modes of transmission, localization within the vector, and their hosts. This comprehensive overview highlights the complex interactions between viruses, their vectors, and the plants they infect, emphasizing the need for targeted strategies in managing these pathogens effectively.

Among all these viruses, a significant proportion are circulative, often responsible for serious diseases that can lead to substantial yield losses. These losses arise from the replication, accumulation, and spread of viral particles within phloem tissues, resulting in a condition known as Phloem Occlusion. This blockage disrupts sap flow, causing an accumulation of carbohydrates in the leaves. Consequently, chloroplasts degenerate, leading to leaf discoloration, reduced chlorophyll content, and inhibited photosynthesis. This manifests as reduced plant height and chlorosis, resembling leaf bleaching due to the loss of chlorophyll. These symptoms make the plant unsuitable for consumption and can significantly impact farmers revenue, often affecting their earnings for over a year.

In France, one of the world's largest cereal producers, several viral diseases linked to circulative viruses threaten crops. The most notable include:

- Wheat Dwarf Virus (WDV):
This disease causes localized infestations that can result in yield losses of up to 70%. In severe cases, the reduction in wheat yields can be substantial, impacting both the quantity and quality of the harvest.

- Wheat Striped Mosaic Virus (WSMV):
Although data are still limited, this disease is increasingly concerning. It is suspected to cause yield losses of about 10 quintals (1,000 kg) per hectare in cases of severe attacks. Wheat mosaics can lead to yield reductions ranging from 20% to 80% for soft wheat and up to 100% for durum wheat.

- Barley Yellow Dwarf Virus (BYDV, MYDV, CYDV):
This is the most well-known and devastating of the diseases. It is recognized as one of the most severe viral diseases, causing significant yield losses, ranging from 5% in cases of low infestation to 90% in the most severe cases. Data from 2012 to 2021 indicate an average loss of 35 quintals per hectare for barley and 17 quintals per hectare for wheat.

Once the plant is infected, we generally witness a reduced height and a chlorosis, similar to a leaf bleach, due to the lack of chlorophyll responsible for the green color of leaves.
These two symptoms make the plant unsuitable for consumption, and can impact the farmers revenue for more than a year.

In the same way, the TSWV (tomato spotted wilt virus) prevents tomatoes to grow if the plants are affected very soon in the growing process.

These viral diseases represent major threats to cereal production in France and world wide, underscoring the urgent need for effective management strategies to protect these crucial crops. One common point between these pathogens is the fact that they are being transmitted from plant-to-plant trough small instects, acting like vectors for the viruses.

B. Plant sensitivity towards viruses
i. Aphids and endangered plantations in France

The vectors of plant viruses play a critical role in the spread of viral diseases within agricultural crops. Among the numerous vectors, aphids stand out as the most significant and influential in the dissemination of viruses. These small insects, belonging to the Aphididae family, feed on plant sap using their stylet—a specialized mouthpart that allows them to pierce plant tissues. When an infected aphid feeds on a plant, it can introduce the virus directly into the host, leading to infection.

Aphids are highly effective vectors due to their rapid reproductive capabilities and their ability to migrate over long distances. For instance, certain aphid species can produce up to 50 new generations per year (Blackman & Eastop, 2000). Their presence is influenced by environmental factors such as temperature and humidity, which can modulate their activity and their ability to spread viruses. In France, from 2010 to 2023, approximately 28% of plants in a given field were infested by aphids, meaning that over a quarter of the plants were at risk of being infected with a virus. This highlights the critical need to implement effective management strategies for aphid populations to mitigate the spread of plant viruses.

ii. Sensitivy timeline for plants

The autumn period is a critical phase for plant sensitivity to aphid-borne viral diseases. During this season, several factors intersect to increase crop vulnerability to viral infections. First, moderate temperatures and relatively high humidity create an environment conducive to aphid survival and activity. These conditions allow aphids to reproduce rapidly and migrate easily between plants, increasing the risk of virus spread.

Plant sensitivity to viral diseases is particularly high before the tillering stage, a critical phase of their development. During this period, young plants are extremely vulnerable to infections, as their defense system is still immature and their rapid growth makes them more receptive to viruses introduced by vectors such as aphids. This increased sensibility is due to several factors: young plants often have softer and less developed tissues, providing easier entry for viruses, and their ability to activate defense mechanisms is still limited.

As plants move from tillering to maturity, a phenomenon called maturity resistance begins to develop. This resistance, which develops gradually, makes plants less sensitive to serious infections. Plants' defense mechanisms become stronger, and they become better able to limit the spread of the virus within their tissues. This transition to increased resistance is a complex phenomenon that depends on many factors, including plant genetics, environmental conditions, and interactions with virus vectors.

It is important to note that plant sensibility does not disappear completely after tillering, but decreases considerably. Later developmental stages benefit from this increased resistance, which limits the development of viral infections and their impact on yield. However, observations must be extended until the onset of winter to ensure effective disease management. Sensitive stages prior to tillering remain critical for monitoring, as infections during this period can have significant impacts on overall crop health and final yields.
In the fall, many cereal crops such as wheat and barley reach their maturation stage. During this phase, young plants and growing crops are particularly vulnerable to viral infections because they do not yet have resistance. Aphids feed on the growing tissues, introducing viruses that can quickly spread throughout the plant. Symptoms of viral infection can then appear later in the season, severely impacting crop yield and quality.

Fight Against Circulative Plant Viruses

A. Natural methods

One effective approach to reducing the risk of virus infestations in crops is through indirect control methods, such as adjusting planting schedules. Specifically, avoiding early sowing can significantly decrease the risk of aphid infestations and subsequent virus transmission. Early-sown crops tend to be more vulnerable as they are exposed to aphids and other vectors during critical growth stages, which heightens the risk of infection. However, this strategy is not universally applicable and may vary depending on regional conditions, such as climate and local pest dynamics. In some areas, other factors may influence the risk, making it necessary to adopt a more tailored, insurance-based management strategy. This approach involves continuously monitoring pest populations and environmental conditions to implement timely interventions that minimize the likelihood of outbreaks, thus ensuring a more resilient crop production system. arva incidence The use of genetically resistant or tolerant plant material is a cornerstone strategy in the fight against viral diseases in crops. Resistant plants have the ability to limit the replication and/or accumulation of viral particles, thereby reducing the severity of the infection. On the other hand, tolerant plants can harbor the virus but do not exhibit severe symptoms, allowing them to maintain yield and quality despite the presence of the pathogen (Cooper and Jones, 1983).

Significant progress has been made in identifying genetic factors that confer tolerance notably in Barley. Among these factors, the Ryd2 gene, located on chromosome 3H, has been particularly effective. This gene enables barley plants to tolerate the presence of BYDV without suffering substantial damage. Ryd2 codes for a protein that is believed to be involved in the plant’s defense mechanisms, although the exact protein and its pathway remain under investigation. Generally, genes like Ryd2 are thought to influence the regulation of cellular processes that control the spread and replication of the virus within the plant. This could involve the activation of signaling pathways that fortify cellular walls or the production of antiviral compounds that inhibit the virus's ability to multiply.

In practical terms, barley plants carrying the Ryd2 gene maintain better growth, grain filling, and overall health even when infected by the virus. This tolerance does not eliminate the virus but ensures that its presence does not significantly impair the plant's physiological functions, particularly photosynthesis and nutrient transport, which are crucial for sustaining yield.

As with barley, significant efforts have also been made in wheat to identify and utilize genetic factors that confer tolerance to viral infections. While wheat has fewer known tolerance genes compared to barley, the identification of the Bdv1 gene in the Brazilian spring wheat variety Frontana marks an important step forward in breeding wheat varieties that can withstand the damaging effects of Barley Yellow Dwarf Virus (BYDV).

Bdv1 likely codes for a protein involved in the plant's defensive response to viral infection. While the precise protein product of Bdv1 is not fully characterized, it is thought to play a role in modulating the plant's immune system, allowing the wheat to better manage and contain the effects of the virus. This could involve triggering pathways that reduce the replication rate of the virus or limit its spread within the plant tissues. The role of Bdv1 is to impart tolerance rather than complete resistance to BYDV-MAV. Wheat plants carrying this gene can still be infected by the virus, but the impact on the plant's growth and yield is significantly lessened. This tolerance ensures that the plants remain productive despite the presence of the virus, which is critical for maintaining crop yields in regions where BYDV is a recurring problem.

While adjusting planting schedules can help reduce the risk of viral infestations by avoiding peak periods of vector activity, and genetic resistance and tolerance have proven to be valuable strategies in managing viral diseases in crops, these methods are not a panacea. The deployment of major resistance genes, such as Ryd2 in barley or Bdv1 in wheat, may exerts selective pressure on pathogens, which can lead to the emergence of resistant viral variants. For example, laboratory studies have shown that, after several successive cycles, viral variants capable of overcoming the resistance conferred by these genes can be selected (Chain et al., 2006). Moreover, the presence of numerous alternative hosts, particularly within the Poaceae family, and the partial resistance of some viruses further complicate the situation in the field. This underscores the necessity for a more integrated approach to disease management. While genetic resistance provides valuable protection, it is not always sufficient to counter the evolving threat of viral pathogens. Consequently, additional measures, including chemical treatments, must be considered to effectively combat these challenges.

B. Chemical methods

Pyrethroids are a significant family of insecticides widely used to control insect vectors of plant viruses and more specifically aphids. These chemicals are characterized by their unique neurotoxic mode of action, classified under Group 3A by the IRAC (Insecticide Resistance Action Committee). Their mechanism involves disrupting sodium channels in the neuronal membranes of insects, leading to overstimulation of the central nervous system. By inhibiting the recovery of sodium channels, pyrethroids cause continuous neuronal firing, resulting in paralysis and death of the insect.

Mode of Action and Application
Pyrethroids primarily act by contact, meaning their effectiveness is directly influenced by the quality of application. An adequate application volume is crucial to ensure that the leaves and other parts of the plant where insects are present are properly treated. Therefore, adhering to specific application volume recommendations is essential to optimize insect control.

Positioning and Timing
For effective treatment, it is advised to apply pyrethroids based on the actual presence of insect vectors rather than solely on plant development stages. Regular monitoring of plants is necessary to determine the optimal timing for treatment.

Persistence of Action
The action of pyrethroids is relatively short-lived, typically lasting 10 to 15 days on treated leaves. It is important to note that new leaves emerging after treatment are not protected, as pyrethroids do not have systemic action. Consequently, additional applications may be needed to protect new growth and maintain effective control over vector populations.

In summary, while pyrethroids are powerful tools in managing insect infestations, their use must be carefully planned and adjusted according to specific crop conditions to ensure maximum effectiveness and sustainable vector management.

In June 2011, the United Kingdom reported notable failures in the efficacy of pyrethroid treatments, a concerning development in the management of insect pests. A key factor contributing to these failures is the presence of genetic mutations conferring resistance to pyrethroids. Specifically, the mutation known as kdr (knockdown resistance) was detected, leading to a resistance rate of approximately 40% in affected populations (Foster, 2014). The kdr mutation affects the sodium channels in the nervous system of insects. Pyrethroids exert their insecticidal effect by binding to these sodium channels, disrupting their normal function, and causing continuous neuronal activity, which ultimately leads to paralysis and death. The kdr mutation alters the structure of these sodium channels, reducing the binding efficiency of pyrethroids and thereby diminishing their neurotoxic effects. In insects with this mutation, the channels are less susceptible to the action of pyrethroids, leading to reduced efficacy of these insecticides. Among the various insect species, the resistance has been prominently observed in a specific clone of the Sitobion avenae aphid population, known as clone SA3. This clone is heterozygous for the kdr mutation, meaning it possesses one resistant allele and one susceptible allele. This genetic configuration allows the aphid to survive exposure to pyrethroids more effectively than other clones without the mutation. The presence of the kdr mutation has been detected not only in the UK but also in other European countries, indicating a broader issue of pyrethroid resistance in the region.

Notable examples include:
- France : Resistance to pyrethroids has been documented in populations of the *Sitobion avenae aphid. Studies have shown that resistance levels can be significant, impacting the effectiveness of pyrethroid-based insecticides in managing aphid populations (Foster, 2014).

- Germany : Research has indicated the presence of kdr mutations in Myzus persicae (the green peach aphid), a major pest affecting multiple crops. This mutation has led to reduced efficacy of pyrethroid treatments, complicating pest control efforts (Vidal et al., 2012).

- Italy : Resistance issues have been observed in various aphid species, including Aphis gossypii (the cotton aphid). These resistance cases are attributed to genetic mutations that decrease the effectiveness of pyrethroid insecticides (Giacomini et al., 2013).

- Spain : Studies have revealed that resistance to pyrethroids is present in *Schizaphis graminum* (the greenbug). The development of resistance in this species has been linked to the kdr mutation, affecting the control of this pest in cereal crops (Morales et al., 2015).

Given the widespread resistance to pyrethroids observed across Europe, there has been a substantial increase in research efforts aimed at understanding and overcoming these resistance mechanisms. and these research into pyrethroid resistance lead to the identification of new resistance mutations. In 2021, a notable advancement was made with the discovery of the SKDR (Sodium Channel Knockdown Resistance) mutation, specifically the M918L mutation, in China. This mutation was first identified by Wang et al. (2020) and represents a critical point in understanding pyrethroid resistance mechanisms.

The SKDR mutation, M918L, is a variant of the kdr (knockdown resistance) mutation, which affects the sodium channels in insects. Sodium channels are essential for nerve signal transmission, and pyrethroids function by disrupting these channels, leading to neurotoxic effects that ultimately kill the insects. The M918L mutation alters the structure of these sodium channels, reducing the binding efficacy of pyrethroids and thereby decreasing their neurotoxic impact on the pests. This discovery of the M918L mutation underscores the evolving nature of resistance in pest populations. The presence of such mutations complicates the management of pyrethroid-resistant insects, as traditional insecticides become less effective. As resistance mechanisms continue to evolve, it is crucial to develop and implement integrated pest management strategies that include monitoring for these mutations, rotating insecticide classes, and employing alternative control methods to mitigate the impact of resistance on agricultural productivity.

The escalating resistance of pests to pyrethroids, as evidenced by various studies across Europe, has highlighted the growing inadequacy of chemical treatments alone in managing agricultural pests. This challenge is compounded by the broader implications of chemical pesticide use, which extend beyond the efficacy of these products. The detrimental effects on biodiversity and human health underscore the urgent need for a more sustainable approach to pest management.

Pyrethroids, while effective in controlling a wide range of pests, have been associated with significant environmental and health concerns. These chemicals, which act as neurotoxins, are known to disrupt the nervous systems of insects by interfering with sodium channels. However, their impact is not confined to pests alone. Research has shown that pyrethroids can also have adverse effects on non-target species, including beneficial insects, such as pollinators and natural predators, which are crucial for maintaining ecological balance. For instance, studies have documented declines in honeybee populations, which are vital for crop pollination, as a result of pesticide exposure (Goulson et al., 2015).

In addition to their ecological impact, pyrethroids pose serious risks to human health. Chronic exposure to these chemicals has been linked to a range of health issues, including neurological disorders, reproductive problems, and developmental effects in children. The U.S. Environmental Protection Agency (EPA) has noted that long-term exposure to pyrethroids can lead to adverse effects such as respiratory problems and skin irritation (EPA, 2020). The risk is further exacerbated for agricultural workers and communities living near treated fields, who are at higher risk of exposure due to their proximity to pesticide applications.

The environmental footprint of pyrethroid use extends beyond immediate health effects. These chemicals can persist in soil and water, leading to long-term contamination and potential harm to aquatic ecosystems. For example, pyrethroids have been found to accumulate in water bodies, where they can negatively affect aquatic life by disrupting reproductive processes and impairing development (Gauthier et al., 2016). Given these significant concerns, it is clear that reliance on chemical pesticides alone is not a viable long-term solution. The combined pressures of pesticide resistance, environmental degradation, and health risks call for a shift towards more sustainable pest management practices.

Moneywise, the estimates of damage related losses go up, at least, 30 billion $USDeach year, hence the abusive use of pesticides from farmers.^[6]
However, pesticides are far from being the most suitable solution in that case, as they pollute soil, water, vegetation, tend to unbalance ecosystems and represent a danger towards human health. As mentionned by the EEA (European Environment Agency), pesticides ingestion through human consumption are responsible for neurological disorders, developmental delays and, for the most part, cancers.

Pesticide use in agriculture often undergo law changes, and can be reauthorized depending on yields losses, partly due to plant viruses. But, as human beings, we're in desperate need for a new solution, as these dangerous chemicals remain stable in the environment, and harmful after being sprayed on plantations.

This is the reason why we want to offer a more targeted & eco-friendly alternative to regular pesticides.

Our selective approach

Our approach addresses these pressing concerns by adopting a targeted method to combat the spread of plant viruses while mitigating the environmental and health impacts associated with traditional pesticides. Our strategy focuses on selectively eliminating the insect vectors responsible for transmitting these viruses, ensuring that our solution is both effective and environmentally friendly.

To achieve this, we plan to engineer a custom bacterium capable of expressing receptors specifically designed to bind to the viral coat proteins of the pathogens of concern. When this engineered bacterium interacts with the virus within the aphid's microbiota, a specific recognition mechanism triggers a signaling pathway that results in the production of a lethal toxin. This toxin effectively targets and neutralizes the aphids, thereby preventing them from transmitting the virus to other plants.

Our project draws inspiration from the innovative ARBO-BLOCK project developed by the iGEM team in Aix-Marseille. This pioneering work provided a foundation for our model, incorporating the concept of using genetically engineered bacteria to disrupt the life cycle of virus-carrying insects. Building on this legacy, our team at Aix-Marseille aims to refine and adapt this model, leveraging our expertise to create a tailored solution for controlling vector-borne plant viruses.

By implementing our model, we aim to address a wide range of vector-borne viruses with greater precision and efficacy. This approach offers a promising alternative to conventional pesticide use, reducing the harmful environmental impacts while maintaining effective virus control. Ultimately, our goal is to enhance crop yields and safeguard human health by providing a sustainable and targeted solution for managing plant viruses.

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DESIGNS

EXPERIMENTS

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References

iGEM Aix-Marseille 2021: Arbo-Block