Safety

The World Health Organization (2021) defines DURC as life-science research findings intended for peaceful and beneficial purposes that can be misused to cause harm to humans, animals, plants, or the environment.

Here, we want to evaluate whether our project presents potential DURC issues specifically.

Our aim is to use gene-specific siRNAs encapsulated in chitosan nanoparticles to control Phytophthora capsici, the pathogen responsible for foot rot disease in black pepper. This approach is designed to be efficient and provide a sustainable solution for farmers. The modularity of the system allows the siRNA sequence to be adapted to target other P. capsici genes or even other plant pathogens, enabling broad agricultural applications.

However, these strengths also introduce potential misuse risks. The ability to alter the siRNA sequence means that this technology could be misused to target key plant genes, leading to widespread crop damage. Such misuse could have serious consequences for food security and the agricultural stability of the country.

While our work is intended solely for agricultural benefit and disease control in black pepper, we acknowledge the importance of implementing safeguards. This includes responsible sequence design and biosafety to minimize the risk of misuse.

Chitosan nanoparticles are a promising and sustainable choice as they are completely biodegradable, reducing the environmental footprint. They enhance the levels of auxins and strigolactones in plant roots, which promotes better colonization by arbuscular mycorrhizal fungi (AMF). This effect has been observed not only in wheat but also in tomato and maize plants (Saleh et al., 2023). Consequently, it may be regarded as a sustainable alternative, as its application is environmentally compatible and does not exert detrimental effects on soil quality or overall ecosystem health. Chitosan can undergo complete degradation within 7 days (Oberlintner et al., 2021).

Chitosan is susceptible to digestion by chitosanase, lysozyme, and exo-β-D-glucosaminidase, each exhibiting different substrate specificity. Chitosanase contributes significantly to the degradation of chitosan nanoparticles and is naturally produced in pepper soil by organisms such as Streptomyces griseus, Bacillus amyloliquefaciens, cyanobacteria, and fungi (Bhuvanachandra et al., 2021; Anju et al., 2023; Yun et al., 2005; Anusree et al., 2019; Thadathil et al., 2014). It specifically targets sequences of three consecutive deacetylated units within the chitosan structure, effectively breaking down the polymer at these sites.

In contrast, lysozyme targets N-acetylated glucosamine, cleaving the chitosan chain at strategic points to facilitate degradation (Mao et al., 2001). Meanwhile, exo-β-D-glucosaminidase cleaves the chitosan chain from the non-reducing end. These enzymes may have different specificities for various forms and degrees of acetylation of chitin and chitosan.

The non-specific chitosan-hydrolyzing enzymes include carbohydrases, proteases, lipases, etc. (Thadathil et al., 2014).

Chitosan degradation produces non-toxic amino sugars. Among these, glucosamine is known for its excellent biocompatibility and safety profile. In humans, glucosamine has been shown to cause neither toxicity nor significant alterations in blood glucose concentration, making it a safe byproduct of enzymatic degradation (Chen et al., 2010). Another important degradation product is N-acetylglucosamine, which is a naturally occurring monosaccharide that plays a vital role in the structural and functional properties of many biological systems. It is abundantly found on the surface of multicellular organisms and serves as a key component of the peptidoglycan layer in bacterial cell walls. It is crucial even for fungal cell wall formation, contributing to chitin synthesis, thereby proving its essential role in different biological domains (Konopka, 2012). These end products not only show that chitosan is completely biodegradable but also highlight its safety. Additionally, the natural and non-toxic nature of the products makes chitosan an excellent choice for sustainable use in medicine and the environment.

We ensured the safety and specificity of our siRNA by prioritizing the evaluation of potential off-targets. The NCBI BLAST tool was used to find the sequence identity and similarity scores, enabling the identification and exclusion of siRNA candidates with significant homology to non-target genes. The remaining candidates were further analyzed using GESS (Genome-wide Enrichment of Seed Sequence matches). By combining these two approaches, the risk of unintended gene silencing was significantly reduced, and the overall safety of the siRNA design was improved.

Ali, G., Sharma, M., Salama, E., Ling, Z., & Li, X. (2022). Applications of chitin and chitosan as natural biopolymer: potential sources, pretreatments, and degradation pathways. Biomass Conversion and Biorefinery, 14(4), 4567–4581. https://doi.org/10.1007/s13399-022-02684-x

Anju AB, Natarajan C, Preetha R, Rajan SA, Soumya VI, Anith KN(2023). Bacterization with Endospore-forming Bacillus spp. Promotes Plant Growth and Suppresses Foot Rot Disease in Black Pepper (Piper nigrum L.) in the Nursery. J Pure Appl Microbiol. 2023;17(2):768-779. https://doi.org/10.22207/jpam.17.2.0

Anusree, T., Suseela Bhai, R., Ahammed Shabeer, T. P., & Oulkar, D. (2019). Streptomyces spp. from Black Pepper Rhizosphere: A Boundless Reservoir of Antimicrobial and Growth Promoting Metabolites. Journal of Biologically Active Products from Nature, 9(1), 1–23. https://doi.org/10.1080/22311866.2018.1561327

Bhuvanachandra, B., Sivaramakrishna, D., Alim, S., Preethiba, G., Rambabu, S., Swamy, M.J., & Podile, A.R. (2021). New Class of Chitosanase from Bacillus amyloliquefaciens for the Generation of Chitooligosaccharides. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.0c05078

Chen, J., Shen, C., & Liu, C. (2010). N-Acetylglucosamine: production and applications. Marine Drugs, 8(9), 2493–2516. https://doi.org/10.3390/md8092493

Fukamizo, T., & Brzezinski, R. (1997). Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function. Biochemistry and Cell Biology, 75(6), 687–696. https://doi.org/10.1139/o97-079

Konopka, J. B. (2012). N-Acetylglucosamine functions in cell signaling. Scientifica, 2012, 1–15. https://pmc.ncbi.nlm.nih.gov/articles/PMC3551598/

Mao, H., Roy, K., Troung-Le, V. L., Janes, K. A., Lin, K. Y., Wang, Y., August, J., & Leong, K. W. (2001). Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. Journal of Controlled Release, 70(3), 399–421. https://doi.org/10.1016/S0168-3659(00)00361-8

Oberlintner, A., Bajić, M., Kalčíková, G., Likozar, B., & Novak, U. (2021). Biodegradability study of active chitosan biopolymer films enriched with Quercus polyphenol extract in different soil types. Environmental Technology & Innovation, 21, 101318. https://doi.org/10.1016/j.eti.2020.101318

Saleh, A. M., El-Soud, W. M. A., Alotaibi, M. O., Beemster, G. T., Mohammed, A. E., & AbdElgawad, H. (2023). Chitosan nanoparticles support the impact of arbuscular mycorrhizae fungi on growth and sugar metabolism of wheat crop. International Journal of Biological Macromolecules, 235, 123806. https://doi.org/10.1016/j.ijbiomac.2023.123806

Thadathil, N., & Velappan, S. P. (2014). Recent developments in chitosanase research and its biotechnological applications: A review. Food Chemistry, 150, 392-399. https://doi.org/10.1016/j.foodchem.2013.10.083

Yun, C., Amakata, D., Matsuo, Y., Matsuda, H., & Kawamukai, M. (2005). New chitosan-degrading strains that produce chitosanases similar to ChoA of Mitsuaria chitosanitabida. Applied and Environmental Microbiology, 71(9), 5138–5144. https://doi.org/10.1128/AEM.71.9.5138-5144.2005