The world’s population is estimated to exceed 10 billion by 2050, and the total global demand for food is expected to increase from 35% in 2010 to 56% in 2050. Insect-pest damage poses a serious threat to the world’s crop productivity causing losses of up to 20–40% of annual crop production worldwide, which corresponds to an estimated value of $220 billion (Saakre et al., 2023).
S. litura (Figure. 1) is an omnivorous and bulimic agricultural pest that greatly affects crop yields due to their wide host range, high reproductive potential, miscellaneous larval feeding, and high food intake (Ramaiah and Maheswari, 2018).
Indiscriminate use of chemical pesticides not only leads to the evolution of insecticide resistance but also causes negative impacts on the environment and toxicity to non-target organisms (Li et al., 2024). Although transgenic crops expressing Bacillus thuringiensis (Bt) genes could reduce the burden of S. litura without harming the environment or human health, the efficacy of Bt crops has attenuated due to the emergence of field-evolved resistant insect pests (Tabashnik et al., 2023). These pose a vital need for development of alternative strategies for the management of S. litura. One such promising strategy is RNA interference (RNAi) technology.
Figure. 1 Life cycle of Spodoptera litura (Ramaiah and Maheswari, 2018).
This year, the Hubu-China team is exploring ways to solve this problem from the perspective of synthetic biology. RNA interference (RNAi) has recently proven to be a useful tool for the control of a variety of pathogens. RNAi can reduce the expression of pest genes that are critical to growth/development or infection processes. This can have a detrimental effect on pathogens.
In this project, we attempted to expressing artificial microRNA (amiRNA) targeting the essential gene of S. litura from the genome of tobacco plastid. To protect the degradation of amiRNA caused by highly active nuclease in S. litura, amiRNA was delivered by a bacteriophage MS2 virus-like particle (VLP; Fig. 3)-based delivery system (Wang et al., 2016).
Figure 3: The schematic diagram of construction and preparation of 2MS-TAT-miR122 VLPs.
RNAi is a posttranscriptional gene-silencing mechanism which is triggered by the introduction of double-stranded RNA (dsRNA) into a eukaryotic cell. The first application of RNAi for pest insect control involved transgenic tobacco and Arabidopsis plants expressing dsRNA that inhibited expression of a P450 monooxygenase gene of Helicoverpa armigera and thereby weakened the insect’s tolerance to gossypol (Mao et al., 2007). Thereafter, the plant-mediated RNAi method is wildly deployed to manage various insect pests (Chung et al., 2021; Saakre et al., 2023).
Currently, plant-mediated RNAi for pest control is mainly based on nuclear transformation. Nevertheless, a large part of long dsRNA transcribed in the nucleus are inevitably cleaved into siRNAs by the plant Dicer‐like (DCL) proteins, thus affecting the RNAi response in insects (Figure. 4).
Figure. 4 Schematic model of plant-mediated RNAi for pest management.
The dsRNAs expressed from nuclear genome are cleaved into siRNAs by the plant’s own RNAi machinery, thus impeding the RNAi responses in some insects, whereas intact dsRNAs can be accumulated in plastid-transformed plants, due to the absence of Dicer proteins in plastids (Li et al., 2023). When ingested by insects, the plant-generated dsRNAs are processed into a number of siRNAs by Dicer, and then assembled with Argonaute proteins (AGOs) into the RNA-induced silencing complex (RISC), which targets and degrades specific mRNAs. Great amounts of dsRNAs produced in plastids can cause high mortality in coleopterans. By contrast, highly active dsRNases in the midgut of lepidopterans can rapidly degraded plastid-expressed dsRNAs, thus attenuating RNAi responses (Li et al., 2023).
To avoid the cleavage of dsRNA produced in plants, RNAi constructs can be expressed in plastids (chloroplasts), which have extremely high copy numbers of plastid genome (e.g., 10, 000 copies in tobacco leaf mesophyll cell) and lack an RNAi machinery, thus allowing high-level accumulation of unprocessed dsRNA (Fig. 2). It has been demonstrated that long dsRNA is taken up more efficiently than siRNA by some coleopteran insect cells (Bolognesi et al., 2012; He et al., 2020; Li et al., 2015), and plastid-expressed dsRNA is able to trigger strong RNAi responses in coleopteran insects, such as Henosepilachna vigintioctopunctata, Leptinotarsa decemlineata and Plagiodera versicolora (Table 1; Li et al., 2023). In addition, the plastid genomes are maternally inherited in most species, thus largely excluding unwanted pollen transmission of transgenes. Therefore, plastid could be applied as an ideal synthetic biology chassis for pest management.
Table 1Recent examples of PM-RNAi in control of arthropod pests.
| Taxonomy | Species | Silenced gene | Host plant | Phenotypic effect | Reference |
| Coleoptera | Henosepilachna vigintioctopunctata | Actin | Potato | Increased mortality | Xu et al., 2023 |
| Leptinotarsa decemlineata | Actin | Potato | Increased mortality | Zhang et al., 2015 | |
| L. decemlineata | v-ATPaseA | Tomato | Down-regulation of target gene | Kaplanoglu et al., 2022 | |
| Plagiodera versicolora. | Actin Srp54k | Poplar | Enhanced mortality | Zhang et al., 2024) | |
| Hemiptera | Bemisia tabaci | Actin | Tobacco | No effect | Dong et al., 2020 |
| Halyomorpha halys | v-ATPaseA | Tomato | Down-regulation of target gene | Kaplanoglu et al., 2022 | |
| Myzus persicae | Heavy chain of dynein | Tobacco | Increased mortality and reduced fecundity | Dong et al., 2022 | |
| Phenacoccus madeirensis | v-ATPaseA | Tomato | Down-regulation of target gene | Kaplanoglu et al., 2022 | |
| Lepidoptera | Helicoverpa armigera | Acetylcholinesterase | Tobacco | Reduced larval weight | Bally et al., 2016 |
| v-ATPaseH | Tobacco | No effect | Fu et al., 2022 | ||
| H. zea | v-ATPaseA, Chitin synthase, Cytochrome P450 monooxygenase | Tobacco | Reduced larval weight | Jin et al., 2015 | |
| Manduca sexta | v-ATPaseA | Tobacco | No effect | Burke et al., 2019 | |
| Thysanoptera | Frankliniella occidentalis | ACT, Tubulin, v-ATPaseB, Snf7 | Tobacco | Increased mortality | Wu et al., 2022 |
| Acari | Tetranychus cinnabarinus | Actin | Tomato | Increased mortality | Wu et al., 2023 |
| T. evansi | Actin | Tomato | Increased mortality | Wu et al., 2023 | |
| T. truncatus | Actin | Tomato | Increased mortality | Wu et al., 2022 |
However, long dsRNA appears to be not an effective insecticidal RNAi molecule against lepidopterans. Fu et al. (2022) showed that tobacco plastid-expressed dsRNA targeting various genes, including β-actin, COPI β, V-ATPaseA, V-ATPaseD and V-ATPaseH, did not initiate effective RNAi responses in H. armigera, whereas the target gene was only reduced in H. armigera fed with nuclear-transformed plants (Fu et al., 2022). These findings were further verified by RNA degradation assays in which plastid-expressed dsRNAs were degraded more rapidly than plant-derived siRNAs when incubated with intestinal fluid of H. armigera (Fu et al., 2022).
MS2 VLPs harboring specific RNA fragments possess favorable features, such as stability, biocompatibility, biodegradability, and suitable molecular size, making them appropriate miRNA vectors.
HIV TAT was the first reported cell penetrating peptide. The regions of TAT 47-57 belong to the protein transduction domain and possess powerful, nontoxic, and efficient transport capacity, which had been clearly confirmed. Hence, TAT 47-57 was selected and designed to display on the surface of MS2 VLP.
Therefore, a novel delivery system based on the surface display of TAT peptides on the surface of MS2 VLPs was established, which can effectively penetrate the cell membrane and suppress genes.
Bolognesi R, Ramaseshadri P, Anderson J, Bachman P, Clinton W, Flannagan R, et al. 2012. Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte). PLoS One 7, e47534.
Chung SH, Feng H, Jander G. 2021. Engineering pest tolerance through plant-mediated RNA interference. Current Opinion in Plant Biology 60, 102029.
Fu J, Xu S, Lu H, Li F, Li S, Chang L, et al. 2022. Resistance to RNA interference by plant-derived double-stranded RNAs but not plant-derived short interfering RNAs in Helicoverpa armigera. Plant, Cell & Environment 45, 1930–1941.
He W, Xu W, Xu L, Fu K, Guo W, Bock R, et al. 2020. Length-dependent accumulation of double-stranded RNAs in plastids affects RNA interference efficiency in the Colorado potato beetle. Journal of Experimental Botany 71, 2670–2677.
Kumar M, Panwar V, Chaudhary V, Kumar R. 2024. Artificial miRNAs: A potential tool for genetic improvement of horticultural crops. Scientia Horticulturae 331, 113160.
Li H, Khajuria C, Rangasamy M, Gandra P, Fitter M, Geng C, et al. 2015. Long dsRNA but not siRNA initiates RNAi in western corn rootworm larvae and adults. Journal of Applied Entomology 139, 432–445.
Li S, Kim D, Zhang J. 2023. Plastid-mediated RNA interference: a potential strategy for efficient insect pest control. Plant, Cell & Environment 46, 2595–2605.
Li W, Yang W, Shi Y, Yang X, Liu S, Liao X, et al. 2024. Comprehensive analysis of the overexpressed cytochrome P450-based insecticide resistance mechanism in Spodoptera litura. Journal of Hazardous Materials 461, 132605.
Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY, Wang LJ, et al. 2007. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nature Biotechnology 25, 1307–1313.
Ramaiah M, Maheswari T. 2018. Biology studies of tobacco caterpillar Spodoptera litura Fabricius. Journal of Entomology and Zoology Studies 6, 2284–2289
Saakre M, Jaiswal S, Rathinam M, Raman KV, Tilgam J, Paul K, et al. 2023. A host-delivered RNA interference for durable pest resistance in plants: advanced methods, challenges, and applications. Molecular Biotechnology 66, 1786–1805.
Tabashnik BE, Fabrick JA, Carrière Y, Wu Y. 2023. Global patterns of insect resistance to transgenic Bt crops: the first 25 years. Journal of Economic Entomology 116, 297–309.
Wang G, Jia T, Xu X, Chang L, Zhang R, Fu Y, et al. 2016. Novel miR-122 delivery system based on MS2 virus like particle surface displaying cell-penetrating peptide TAT for hepatocellular carcinoma. Oncotarget 7, 59402–59416.
VLP stands for virus-like particles. Virus-like particles are
molecules that mimic viruses but are not infectious. They are a very
effective way of creating vaccines against diseases such as human
papillomavirus (HPV), hepatitis B, malaria, and more.
Artificial microRNA (amiRNA) refers to RNA molecules designed to
mimic primary miRNA stem-loops, with sequences tailored for
specific target transcripts while retaining recognition sequences
for cleavage. They offer a safer alternative to shRNAs in gene
silencing applications, demonstrating improved safety profiles
without compromising efficacy.
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that
mediates resistance to both endogenous parasitic and exogenous
pathogenic nucleic acids, and regulates the expression of
protein-coding genes. This natural mechanism for sequence-specific gene
silencing
promises to revolutionize experimental biology and may have
important practical applications in functional genomics,
therapeutic intervention, agriculture and other areas.
Plastid-mediated RNA interference (PM-RNAi) has emerged as a promising strategy for pest control.Expression from the plastid genome of stable double-stranded RNAs (dsRNAs) targeted against essential insect genes can effectively control some herbivorous beetles.
Spodoptera litura, otherwise known as the tobacco cutworm or
cotton leafworm, is a nocturnal moth in the family Noctuidae. S.
litura is a serious polyphagous pest in Asia, Oceania, and the
Indian subcontinent that was first described by Johan Christian
Fabricius in 1775. Its common names reference two of the most
frequent host plants of the moth.
The measure of the efficiency and effectiveness with which agricultural inputs (such as land, labor, capital, and technology) are used to produce agricultural outputs. It is often expressed in terms of yield per unit area or per unit of input. Agricultural productivity can be affected by a range of factors, including climate, soil quality, farming practices, and the use of fertilizers and pesticides.
External factors that can cause a response or change in an organism's behavior or physiology. These stimuli can include abiotic factors such as temperature, light, water, and atmospheric gases, as well as biotic factors like interactions with other organisms, including predators, competitors, and pathogens. In agriculture, environmental stimuli can affect plant growth, development, and crop yields, as well as the activity and survival of pests and beneficial organisms.
Organisms that can cause damage or disease to plants, animals, or humans. Pests typically refer to insects, mites, nematodes, and other animals that feed on crops or stored products, while pathogens are microorganisms, such as bacteria, viruses, fungi, and oomycetes, that cause diseases. Both can significantly reduce agricultural productivity and have economic, social, and ecological impacts.
A specialized agency of the United Nations that leads international efforts to defeat hunger and improve nutrition and food security. The FAO provides technical assistance, policy advice, and knowledge exchange to help countries make informed decisions about agriculture, forestry, fisheries, and rural development. It also monitors global food supplies and works to prevent and mitigate food crises.
Substances designed to kill, repel, or control insect populations. They may act through various mechanisms, such as disrupting the nervous system, inhibiting growth, or preventing reproduction. Chemical insecticides have been widely used in agriculture for pest control, but their overuse has led to issues such as resistance, environmental contamination, and negative effects on non-target organisms, including pollinators and natural enemies of pests.
The ability of an insect population to survive and reproduce after exposure to a pesticide that would normally be lethal. Resistance develops due to genetic changes within the population, often as a result of selection pressure from repeated applications of the same or similar chemicals. Over time, the resistant individuals become more common, leading to a decrease in the efficacy of the pesticide.
An order of insects that includes butterflies and moths. Lepidopterans are characterized by large, scale-covered wings, a coiled proboscis for feeding, and a complete metamorphosis life cycle. Many species are important pollinators, but some, particularly certain moth larvae, are significant agricultural pests.
A cellular mechanism by which small RNA molecules, such as small interfering RNAs (siRNAs) and microRNAs (miRNAs), silence gene expression. This process can occur at the transcriptional level, where it affects DNA methylation and chromatin structure, or at the post-transcriptional level, where it leads to the degradation of mRNA or blocks its translation into proteins. RNAi is a powerful tool for studying gene function and has potential applications in pest control, therapy, and functional genomics.
An enzyme that catalyzes the polymerization of N-acetylglucosamine units to form chitin, a long-chain polysaccharide that is a major component of the exoskeleton in arthropods, including insects, and the cell walls of fungi. Chitin synthases are essential for the development and integrity of these structures, making them attractive targets for pest control strategies.
A type of small RNA molecule that is engineered to mimic the structure and function of endogenous microRNAs. AmiRNAs can be designed to target specific genes for downregulation through the RNA interference pathway. By binding to complementary sequences in the target mRNA, amiRNAs can inhibit gene expression, leading to reduced levels of the corresponding protein.
A structural protein that forms the capsid, or outer shell, of the bacteriophage MS2. The MS2 CP self-assembles into icosahedral particles that can encapsulate and protect RNA. When used in synthetic biology, the MS2 CP can be modified to display peptides or other molecules on the surface of the virus-like particles (VLPs) it forms, allowing for targeted delivery of RNA to cells.
The genetic modification of plastids, such as chloroplasts, which are organelles found in plant cells responsible for photosynthesis. Plastid engineering allows for the introduction of foreign genes into the plastid genome, resulting in high-level expression of the introduced genes, stable inheritance, and the potential for enhanced biosafety, since plastid genomes are generally not transferred through pollen.
Plants that have been genetically modified by introducing foreign DNA into their plastid genomes. Transplastomic plants can express the introduced genes at very high levels, and the genetic modifications are usually maternally inherited, reducing the risk of gene flow to wild relatives. This approach can be used to confer traits such as herbicide tolerance, pathogen resistance, or the production of pharmaceuticals and industrial compounds.
Changes in gene function that do not involve alterations to the underlying DNA sequence. Epigenetic mechanisms, such as DNA methylation, histone modification, and chromatin remodeling, can influence how genes are expressed. In the context of genetic engineering, epigenetic effects can lead to unintended silencing or activation of genes, which may affect the stability and predictability of the engineered traits.
Short, double-stranded RNA molecules, typically 20-25 base pairs in length, that are involved in the RNA interference (RNAi) pathway. siRNAs can be generated from longer dsRNA precursors and guide the RNA-induced silencing complex (RISC) to cleave or repress the expression of complementary mRNA, thereby silencing the target gene. siRNAs are used in research and have therapeutic potential for treating diseases caused by aberrant gene expression.