Background

In agriculture, maximizing crop yields has been the supreme imperative since the beginning of agrarian societies. Pesticides play a crucial role in the increase in crop production. The use of pesticides has led to substantial increases in yields for many crops and has greatly benefited farmers economically [1] . It is estimated that without the use of pesticides, fruit production would decrease by 78%, vegetables by 54%, and grains by 32% [2] .

Limitations of chemical pesticides

Currently, chemical pesticides are the mainstream; however, they have several limitations. Traditional chemical pesticides pose challenges that degrade the production environment for farmers, including the increase of resistant bacteria and pests, as well as potential negative impacts on the environment and human health with prolonged use. These issues severely undermine the sustainablity of agriculture. Additionally, difficult-to-control pests and diseases that cannot be managed by existing control systems are also problems to solve.

On the other hand, the current circumstances of developing chemical pesticides has become stringent. Developing pesticides requires over ten years and costs ranging from tens of millions to hundreds of millions of dollars. Furthermore, global regulations on environmental impact and food safety have been strengthened, and as safety-related data for registration increases, development costs are also rising. This situation limits pesticide development to large manufacturers, and developers tend to focus on highly profitable targets to recover the substantial development costs.

RNA pesticides

RNA pesticides have the potential to overcome these limitations of existing chemical pesticides. RNA pesticides utilize RNA interference (RNAi) and leverage conserved pathways in eukaryotes to regulate gene expression post-transcriptionally by targeting and degrading specific messenger RNA (mRNA) or inhibiting their translation [8] [9] .

RNA pesticides can achieve effectiveness by merely altering the target gene sequences, making it easier to avoid resistance in pests and pathogens [10] . Additionally, compared to chemical pesticides, RNA pesticides degrade quickly in the environment and have low residual levels [11] . By specifically targeting the genes of pests and pathogens, RNA pesticides minimize impacts on non-target organisms [11] . Furthermore, RNA pesticides can extend pest and disease control to soil-borne pathogens that traditional pesticides could not manage [11] .

Moreover, RNA pesticides can update their targets at significantly lower costs than chemical pesticides, and once developed, RNA pesticides can substantially reduce the burden of time and expenses.

Challenges of RNA pesticides

To commercialize RNA pesticides, they must be implemented in a way that meets farmers’ demands. Based on our interviews , the following four points were identified as essential for pesticides to satisfy farmers’ needs:

1. Durability

The effect should last for more than one month with a single application.

2. Broad-Spectrum Efficacy

Effective against various pests and diseases.

3. Economic Feasibility

Costs should be maintained at approximately 3–5% of the total agricultural expenses.

4. Safety

Pesticides should have minimal impact on humans and the environment, ensuring they are safe.

In particular, the issue of durability (1) is crucial for the implementation of RNA pesticides. RNA alone decomposes in the natural environment within a maximum of about four days [12] [13] , making it difficult to achieve the month-long effectiveness required by conventional chemical pesticides. Additionally, RNA is easily washed away by rain and wind, making it physically challenging to remain in place.

To address the durability challenge, one of the most effective apporach is to encapsulate RNA in artificial lipid membranes[14] . However, this approach tends to result in high production costs, leading to higher prices for farmers. Academically, methods such as chemically modifying RNA to enhance stability [15] are being researched, but each modification requires registering a new compound for long-term pesticide approval, making rapid responses to needs difficult. These issues are also related to the economic feasibility challenge (3).

Regarding safety (4), RNA pesticides themselves are generally considered safe. However, caution is needed in their formulation and implementation methods. For example, substances used in formulations might have environmental or human impacts.

For broad-spectrum efficacy (2), it is possible to address this by simultaneously applying multiple types of RNA.

Thus, the absence of implementation methods for RNA pesticides that achieve high durability at low costs, have minimal impact on humans and the environment, and are easy to apply is an urgent issue that needs to be resolved for RNA pesticides.

Solution

We propose “Modules for Optimized Viability and Efficacy of RNA Pesticides (MOVE)” to address the challenges of RNA pesticides, durability, broad-spectrum efficacy, economic feasibility, and safety.

MOVE is a RNA pesticide implementation module in which RNA is encapsulated within membrane vesicle (MV) that display proteins on their surface. By encapsulating RNA in MV, the stability of RNA is enhanced by protecting it from environmental RNA-degrading enzymes, thereby increasing durability. Additionally, by displaying functional proteins such as adhesion proteins on the surface of the MV, physical stability is also ensured.

To produce RNA-encapsulated MV at low cost, we utilize Escherichia coli(E. coli) engineered with a Polymer Intracellular Accumulation-triggered system for Membrane Vesicle Production (PIA-MVP) . PIA-MVP can significantly increase MV production through the internal pressure generated by the accumulation of poly(3-hydroxybutyrate) (PHB) produced and stored within E. coli [16] . In this pathway, the production of PHB increases with the concentration of glucose in the medium. Therefore, by adjusting the glucose concentration in the medium, the production volume of MV can be controlled with high flexibility.

By enabling E. coli with PIA-MVP to produce one or more short hairpin RNA (shRNA) that induce RNA interference targeting specific genes, RNA-encapsulated MV are created. This system allows for mass production of MV compared to conventional MV production and leverages the ubiquitous material glucose, enabling scalable and low-cost production of RNA-encapsulated MV in the future.

Furthermore, by enabling E. coli with PIA-MVP to display functional proteins on the MV surface, the produced MV are endowed with these surface-displayed functional proteins. We adopted the SpyCatcher-SpyTag system [17] to display multiple proteins on the same membrane surface. This system naturally forms isopeptide bonds between a short peptide (SpyTag) and a small protein (SpyCatcher).

Specifically, scaffold proteins fused with SpyCatcher and functional proteins fused with SpyTag are simultaneously expressed and displayed on the cell surface. This allows for high modularity, enabling the simultaneous surface display of multiple functional proteins within a single system and making it easy to change the proteins being displayed. This facilitates the presentation of functional proteins to remain longer in specific environments and enhances the formulation aspects of pesticides, such as efficient penetration into target organisms.

You can see the detail project design in the Design page .

Expected Effects

MOVE can address multiple currently concerning issues:

Improved Durability :

Encapsulating RNA in MV protects it from environmental RNA-degrading enzymes, enhancing durability. Additionally, surface-displayed proteins help physically retain the RNA in place, further increasing durability.

Achievement of Broad-Spectrum Efficacy

MOVE is a module that can individually encapsulate various types of RNA. By mixing the modules containing different types of RNA and providing them to farmers as a single pesticide, it can be implemented as a pesticide capable of addressing various pests and diseases.

Cost Reduction

The high productivity of PIA-MVP allows for low-cost enhancement of RNA durability.

Ensured Safety

As MOVE consists of biologically derived modules, it is unlikely to spread or proliferate in the environment, and no genetically modified organisms are released during the use of MOVE. This emphasizes the advantages of RNA pesticides, which have minimal burden on humans and the environment.

Additionally, the following secondary effects of MOVE can be highlighted:

Future Prospects

MOVE builds upon the efforts of previous iGEM teams (e.g., 2023 SZU-China [18] , 2021 Ecuador [19] , 2020 AU_China [20] , 2015 Lethbridge [21] ) that focused on implementing RNA pesticides for individual diseases. We propose a concept of a versatile module to advance RNA pesticides as a whole.

MOVE can solve all the challenges in implementing RNA pesticides, including “durability,” “broad-spectrum efficacy,” “economic feasibility,” and “safety,” thereby strengthening RNA pesticides. This leads to the realization of three aspects of MOVE: constructing a delivery system for RNA pesticides ( MOVE RNA Pesticides ), further advancing current pest and disease control through RNA pesticides ( MOVE Pest Control ), and transforming agriculture for the better ( MOVE Agriculture ).

In the future, we plan to optimize specific production processes, conduct efficacy tests on various organisms, and perform safety evaluations to advance the practical implementation of MOVE. Additionally, we aim to deepen collaborations with farmers and related companies to develop products that meet on-site needs.

Furthermore, regulatory challenges must be considered. Regulations regarding RNA pesticides and biologically derived formulations vary by country and region, requiring safety and environmental impact assessments. It is essential to appropriately conduct these evaluations and develop strategies to obtain legal approvals.

Ultimately, MOVE is expected to contribute to the realization of sustainable agriculture as an innovative solution in the agricultural sector.

References

[1] Jerry Cooper, Hans Dobson, The benefits of pesticides to mankind and the environment . Crop Protection  26(9)  , 1337-1348 (2007).

[2] Tudi M, Daniel Ruan H, Wang L, Lyu J, Sadler R, Connell D, Chu C, Phung DT, Agriculture Development, Pesticide Application and Its Impact on the Environment . Int J Environ Res Public Health  18(3)  , 1112(2021).

[3] Chun-Sen Ma, Wei Zhang, Yu Peng, Fei Zhao, Xiang-Qian Chang, Kun Xing, Liang Zhu, Gang Ma, He-Ping Yang & Volker H. W. Rudolf, Climate warming promotes pesticide resistance through expanding overwintering range of a global pest . Nat Commun  12 , 5351 (2021).

[4] Osaka Prefecture, Department of Environment, Agriculture, Forestry, and Fisheries, Agricultural Policy Office, Promotion Division, Plant Disease and Pest Control Group, “Measures Against Drug-Resistant Bacteria (Gray Mold Disease)”, https://www.jppn.ne.jp/osaka/gijyutu/haikabi/haikabi.html

[5] S. Singh, B. Singh, A. P. Singh, Nematodes: a threat to sustainability of agriculture . Procedia Environmental Sciences 29 , 215–216 (2015).

[6] R. G. Hatfield, J. P. Strange, J. B. Koch, S. Jepsen, I. Stapleton, Neonicotinoid pesticides cause mass fatalities of native bumble bees: a case study from Wilsonville, Oregon, United States . Environmental Entomology 50 , 1095–1104 (2021).

[7] AgbioInvestor, “Time and Cost of New Agrochemical Product Discovery, Development and Registration -A Study on Behalf of Crop Life International-”, Feb. 2024, https://croplife.org/wp-content/uploads/2024/02/Time-and-Cost-To-Market-CP-2024.pdf

[8] Hannon, G. RNA interference . Nature  418 , 244–251 (2002)

[9] Wilson RC, Doudna JA. Molecular mechanisms of RNA interference . Annu Rev Biophys 42 , 217-39 (2013)

[10] L. Shaffer, RNA-based pesticides aim to get around resistance problems. Proceedings of the National Academy of Sciences  117 , 32823–32826 (2020).

[11] Hashiro, Shuhei, Current Status of RNA Pesticides and Expectations for Double-Stranded RNA AnalysisRNA農薬の現状と二本鎖RNA分析への期待 . Journal of the Mass Spectrometry Society of Japan . 71 . 75-78 (2023) .

[12] Christiaens Olivier, Tardajos Myriam G., Martinez Reyna Zarel L., Dash Mamoni, Dubruel Peter, Smagghe Guy, Increased RNAi Efficacy in Spodoptera exigua via the Formulation of dsRNA With Guanylated Polymers , Frontiers in Physiology , 9 (2018).

[13] GreenLight Biosciences, “Products Pipeline”,https://greenlightbiosciences.com/product-pipeline/

[14] Agrospheres, https://www.agrospheres.com/

[15]J. D. Howard, M. Beghyn, N. Dewulf, Y. De Vos, A. Philips, D. Portwood, P. M. Kilby, D. Oliver, W. Maddelein, S. Brown, M. J. Dickman, Chemically modified dsRNA induces RNAi effects in insects in vitro and in vivo: A potential new tool for improving RNA-based plant protection . Journal of Biological Chemistry  298 , 102311 (2022).

[16] S. Koh, M. Sato, K. Yamashina, Y. Usukura, M. Toyofuku, N. Nomura, S. Taguchi, Controllable secretion of multilayer vesicles driven by microbial polymer accumulation . Scientific Reports  12  (2022).

[17] B. Zakeri, J. O. Fierer, E. Celik, E. C. Chittock, U. Schwarz-Linek, V. T. Moy, M. Howarth, Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin . Proceedings of the National Academy of Sciences  109  (2012).

[18] 2023 SZU-China, https://2023.igem.wiki/szu-china

[19] 2021 Ecuador, https://2021.igem.org/Team:Ecuador

[20] 2020 CAU_China, https://2020.igem.org/Team:CAU_China

[21] 2015 Lethbridge, https://2015.igem.org/Team:Lethbridge