Search Results (68)

Exogenously induced RNA interference (exoRNAi) is a powerful biotechnology tool for precise gene regulation. The plant chalcone synthase (CHS) gene serves as a valuable model for molecular biology due to its central role in flavonoid biosynthesis. However, there are currently very few studies addressing the advantages and disadvantages of in vitro (enzymatic) or in vivo (bacterial) methods for producing double-stranded RNA (dsRNA) for exogenous application. This study aims to optimize and compare the two methods for producing dsRNAs targeting the Arabidopsis thaliana CHS gene: enzymatic synthesis in vitro using a commercial kit and bacterial synthesis in vivo using an engineered E. coli HT115 (DE3) system. Bacterial synthesis conditions were optimized with respect to IPTG concentration and cultivation time, and the resulting dsRNA preparations were purified and quality-controlled. Their biological activities were assessed by treating A. thaliana plants and analyzing the effects on AtCHS gene expression and flavonoid production using qRT-PCR and HPLC-MS. The results demonstrated that purified AtCHS-dsRNA from both methods effectively suppressed AtCHS expression and downstream flavonoid biosynthetic gene expression, leading to significant reductions in anthocyanins and flavanols. This study confirmed the efficacy of exogenous dsRNAs in regulating plant metabolic pathways and provided a comparative analysis of dsRNA synthesis methods, highlighting their benefits and limitations for practical applications in plant biology and protection. Full article

Spray-induced gene silencing (SIGS) represents a transformative paradigm in sustainable pest management, utilizing the exogenous application of double-stranded RNA (dsRNA) to achieve sequence-specific silencing of essential genes in arthropod pests. Unlike transgenic approaches, sprayable RNA interference (RNAi) biopesticides offer superior versatility across crop systems, flexible application timing, and a more favorable regulatory and public acceptance profile. The 2023 U.S. EPA registration of Ledprona, the first sprayable dsRNA biopesticide targeting Leptinotarsa decemlineata, marks a significant milestone toward the commercialization of non-transformative RNAi technologies. Despite the milestone, large-scale field deployment faces critical bottlenecks, primarily environmental instability, enzymatic degradation by nucleases, and variable cellular uptake across pest taxa. This review critically analyzes the mechanistic basis of spray-applied RNAi and synthesizes the recent technological breakthroughs designed to overcome physiological and environmental barriers. We highlight advanced delivery strategies, including nuclease inhibitor co-application, liposome encapsulation, and nanomaterial-based formulations that enhance persistence on plant foliage and uptake efficiency. Furthermore, we discuss how innovations in microbial fermentation have drastically reduced synthesis costs, rendering industrial-scale production economically viable. Finally, we outline the roadmap for broad adoption, addressing essential factors such as biosafety assessment, environmental fate, resistance management protocols, and the path toward cost-effective manufacturing. Full article

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi (Syd.), poses a serious threat to global soybean production. The main approach to managing this disease has been through repeated fungicide applications which have reduced efficacy due to fungicide resistance. Recently, spray-induced gene silencing (SIGS) through exogenous application of double-stranded RNA (dsRNA) has emerged as a promising approach for plant disease management. In the present study, twelve different dsRNAs targeting genes important for P. pachyrhizi urediniospore germination, infection of the host plant or resistant to commonly used fungicides were produced in Escherichia coli on a large scale. Nine of these dsRNAs significantly reduced ASR severity (by 24.0% to 81.1%) and fungal biomass (50.5% to 83.1%) compared to the control when applied as a foliar spray in our growth chamber studies. Three of the most effective dsRNAs targeting an acyltransferase (ACE), cytochrome B (CYTB1) and a reductase (S12) also significantly reduced disease severity (78.2 to 82.3%) and fungal growth (79.8 to 85.4%) compared to the control in the greenhouse studies. Further investigation of the P. pachryrhizi urediniospore germination and hyphal growth in the presence of these dsRNAs in vitro revealed these dsRNAs reduced the spore germination rate from 72.1% to 0.0–26.6% at 4.5 h and hyphal growth from 254.0 µm to 2.7–40.5 µm at 9 h, with dsRNA targeting the S12 gene being the most effective. These results highlight the potential of SIGS using selected dsRNAs as a sustainable strategy for managing ASR through suppressing urediniospore germination and hyphal growth. Full article

Efficient delivery of exogenous genetic material remains a core challenge in plant biotechnology, holding profound implications for sustainable agricultural and forestry development. Although traditional delivery methods such as Agrobacterium-mediated transformation, gene gun bombardment, and electroporation have been widely applied in plant genetic engineering, these systems exhibit limitations including species-dependent efficacy, propensity to cause plant tissue damage, low transformation efficiency, susceptibility to environmental factors. In recent years, with the advancement of nanotechnology, nanoparticle-based nucleic acid delivery systems are emerging as novel tools for applications such as novel tools for dsRNA or transgene delivery. These systems leverage the unique physicochemical properties of nanomaterials, including size-dependent phenomena, tunable surface charge, and enhanced membrane penetration capabilities, to achieve targeted delivery and stable expression of genetic payloads. Nevertheless, nanomaterial-mediated gene delivery systems for plants are still in their nascent stages, and their widespread application faces numerous challenges. This article briefly introduces traditional delivery methods, systematically reviews the applications and progress of nanoparticle-based nucleic acid delivery systems, and discusses the cross-species applicability of nanoparticles, as well as the associated biosafety concerns. We aim to offer insights for tackling the prevailing technical bottlenecks and to provide guidance for the rational design of nanomaterials that efficiently traverse the plant cell wall–plasma membrane barrier and stably deliver nucleic acids without eliciting phytotoxicity. Full article

Cucumber green mottle mosaic virus (CGMMV) represents a serious threat in the production of watermelon. Small RNAs facilitate a mechanism known as RNA interference (RNAi), which regulates gene expression. RNAi technology employs foreign double-stranded RNAs (dsRNAs) to target and reduce the expression levels of specific genes in plants by interfering with their mRNAs. In this study, watermelon plants were treated with dsRNAs of CGMMV MET, IR, and HEL fragments that had been generated in E. coli HT115. We investigated variations in several factors, including viral accumulation, virus-derived small interfering RNAs (vsiRNAs), and symptom severity. MET-dsRNA, IR-dsRNA and HEL-dsRNA dramatically decreased the symptoms of CGMMV in plants in the growth chamber test. Plants treated with viral-derived dsRNA showed a considerable decrease in both virus titers and vsiRNA levels. We also explored the mobility of spray-on dsRNA-derived long dsRNA and discovered that it could be identified in both inoculated leaves and the systemic leaves. IR-dsRNA outperformed MET-dsRNA and HEL-dsRNA in dsRNA therapy. Illumina sequencing of small RNAs from watermelon plants treated with IR-dsRNA and those that were not treated showed that the decreased accumulation of vsiRNAs was consistent with interference with CGMMV infection in systemic leaves. dsRNA-treated plants showed a higher level of 24-nt viral siRNA and lower level of 22-nt viral siRNA accumulation, while 22-nt viral siRNA predominated in untreated plants, indicating that dsRNA treatment improved DCL3 activity. In conclusion, our research provides deeper insights into the mechanism of antiviral RNA interference and confirms the effectiveness of applying dsRNA locally to enhance plant antiviral activity. Full article

The invertebrate neuropeptide F (NPF) signaling plays versatile roles in diverse biological activities and processes. Still, whether and how it mediates feeding and digestion in Pomacea canaliculate remain gaps in our knowledge. Herein, we first identified and characterized PcNPFR via bioinformatics analysis in P. canaliculate, which is a polyphagous herbivore with a voracious appetite that causes devastating damages to ecosystem functioning and services in colonized ranges. Double stranded RNA (dsRNA)-based RNA interference (RNAi) and exogenous rescue were utilized to decipher and substantiate underlying mechanisms whereby NPFR executed its modulatory functions. Multiple sequence alignment and phylogeny indicated that PcNPFR harbored typical seven transmembrane domains (7 TMD) and belonged to rhodopsin-like GPCRs, with amino acid sequence sharing 27.61–63.75% homology to orthologues. Spatio-temporal expression profiles revealed the lowest abundance of PcNPFR occurred in pleopod tissues and the egg stage, while it peaked in male snails and testes. Quantitative real-time PCR (qRT-PCR) analysis showed that 4 µg dsNPFR and 10⁻⁶ M trNPF (NPFR agonist) were optimal doses to exert silencing and rescue effects, accordingly with sampling time at 3 days post treatments. Moreover, the dsNPFR injection (4 µg) at 1/3/5/7 day/s delivered silencing efficiency of 32.20–74.01%. After 3 days upon dsNPFR knockdown (4 µg), mRNA levels of ILP7/InR/Akt/PI3Kc/PI3K_R were significantly downregulated compared to dsGFP controls, except FOXO substantially upregulated at both transcript and translation levels. In addition, the activities of alpha-amylase, protease and lipase were significantly suppressed, accompanied by decreased leaf area consumption, attenuated feeding behavior and diminished feeding rate. Moreover, expression trends were opposite and proxies were partially or fully restored to baseline levels post exogenous compensation of trNPF, suggesting phenotypes specifically attributable to PcNPFR RNAi but not off-target effects. PcNPFR is implicated in both feeding and digestion by modulating the ISP pathway and digestive enzyme activities. It may serve as a promising molecular target for RNAi-based antifeedants to manage P. canaliculate invasion. Full article

Contact unmodified antisense DNA biotechnology (CUADb), developed in 2008, employs short antisense DNA oligonucleotides (oligos) as a novel approach to insect pest control. These oligonucleotide-based insecticides target pest mature rRNAs and/or pre-rRNAs and have demonstrated high insecticidal efficacy, particularly against sap-feeding insect pests, which are key vectors of plant DNA viruses and among the most economically damaging herbivorous insects. To further explore the potential of CUADb, this study evaluated the insecticidal efficacy of short 11-mer antisense DNA oligos against Coccus hesperidum, in comparison with long 56-mer single-stranded and double-stranded DNA sequences. The short oligos exhibited higher insecticidal activity. By day 9, the highest mortality rate (97.66 ± 4.04%) was recorded in the Coccus-11 group, while the most effective long sequence was the double-stranded DNA in the dsCoccus-56 group (77.09 ± 6.24%). This study also describes the architecture of the DNA containment (DNAc) mechanism, highlighting the intricate interactions between rRNAs and various types of DNA oligos. During DNAc, the Coccus-11 treatment induced enhanced ribosome biogenesis and ATP production through a metabolic shift from carbohydrates to lipid-based energy synthesis. However, this ultimately led to a ‘kinase disaster’ due to widespread kinase downregulation resulting from insufficient ATP levels. All DNA oligos with high or moderate complementarity to target rRNA initiated hypercompensation, but subsequent substantial rRNA degradation and insect mortality occurred only when the oligo sequence perfectly matched the rRNA. Both short and long oligonucleotide insecticide treatments led to a 3.75–4.25-fold decrease in rRNA levels following hypercompensation, which was likely mediated by a DNA-guided rRNase, such as RNase H1, while crucial enzymes of RNAi (DICER1, Argonaute 2, and DROSHA) were downregulated, indicating fundamental difference in molecular mechanisms of DNAc and RNAi. Consistently, significant upregulation of RNase H1 was detected in the Coccus-11 treatment group. In contrast, treatment with random DNA oligos resulted in only a 2–3-fold rRNA decrease, consistent with the normal rRNA half-life maintained by general ribonucleases. These findings reveal a fundamental new mechanism of rRNA regulation via complementary binding between exogenous unmodified antisense DNA and cellular rRNA. From a practical perspective, this minimalist approach, applying short antisense DNA dissolved in water, offers an effective, eco-friendly and innovative solution for managing sternorrhynchans and other insect pests. The results introduce a promising new concept in crop protection: DNA-programmable insect pest control. Full article

Exogenous RNA application, also known as spray-induced gene silencing (SIGS), is a new approach in plant biotechnology that utilizes RNA interference (RNAi) to modify plant traits. This technique involves applying RNA solutions of double-stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), or microRNA (miRNA) directly onto plant surfaces. This triggers RNAi-mediated silencing of specific genes within the plant or invading pathogens. While extensively studied for enhancing resistance to pathogens, the application of exogenous RNA to regulate plant endogenous genes remains less explored, creating a rich area for further research. This review summarizes and analyzes the studies reporting on the exogenously induced silencing of plant endogenes and transgenes using various RNA types. We also discuss the RNA production and delivery approaches, analyze the uptake and transport of exogenous RNAs, and the mechanism of action. The analysis revealed that SIGS/exoRNAi affects the expression of plant genes, which may contribute to crop improvement and plant gene functional studies. Full article

The insect order Lepidoptera contains many species that are considered to be agricultural pests. Specific double-stranded RNA-degrading enzymes in some moth species decrease the efficiency of RNA interference (RNAi). RNAi refers to the efficient and specific degradation of homologous mRNA induced by highly conserved, double-stranded RNA during evolution. The dsRNase enzymes can specifically recognize exogenous dsRNA, and bind to and degrade dsRNA, resulting in the inability of dsRNA to play its role. Although dsRNases play an important role in dsRNA degradation, there has been limited research on these enzymes. In this study, we successfully identified four genes related to dsRNases (named SeRNase1, SeRNase2, SeRNase3 and SeRNase4) from the genome of Spodoptera exigua. To overcome the rapid degradation of dsRNA in the midgut of S. exigua, we combined nanotechnology with biology and developed a new strategy to administer RNAi to insect pests. This binding block directed contact between the dsRNA and SeRNases to improve the efficiency of RNAi in suppressing gene expression. We demonstrate the potential of using nanotechnology to provide a novel RNAi delivery method for pest control. Full article

The increasing challenges posed by plant viral diseases demand innovative and sustainable management strategies to minimize agricultural losses. Exogenous double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) represents a transformative approach to combat plant viral pathogens without the need for genetic transformation. This review explores the mechanisms underlying dsRNA-induced RNAi, highlighting its ability to silence specific viral genes through small interfering RNAs (siRNAs). Key advancements in dsRNA production, including cost-effective microbial synthesis and in vitro methods, are examined alongside delivery techniques such as spray-induced gene silencing (SIGS) and nanocarrier-based systems. Strategies for enhancing dsRNA stability, including the use of nanomaterials like layered double hydroxide nanosheets and carbon dots, are discussed to address environmental degradation challenges. Practical applications of this technology against various plant viruses and its potential to ensure food security are emphasized. The review also delves into regulatory considerations, risk assessments, and the challenges associated with off-target effects and pathogen resistance. By evaluating both opportunities and limitations, this review underscores the role of exogenous dsRNA as a sustainable solution for achieving viral disease resistance in plants. Full article

Fusarium oxysporum is a widespread soil-borne fungal pathogen that can infect various plants, causing wilt and root rot diseases. The root rot disease of Atractylodes macrocephala caused by F. oxysporum is among the most serious diseases associated with continuous cropping, significantly hindering its sustainable development. In this study, we aimed to investigate the effect of exogenous application of double-stranded RNA (dsRNA) on silencing the F. oxysporum Tup1 gene to reduce its virulence and to evaluate its potential application in controlling root rot disease in A. macrocephala. The Tup1 gene was amplified from the F. oxysporum genome, and different lengths of Tup1-dsRNA were designed and synthesized. The uptake of dsRNA by the fungus was verified using Tup1-dsRNA labeled with fluorescein, and in vitro dsRNA treatment experiments were conducted to assess its impact on the growth and virulence of F. oxysporum. Additionally, Tup1-dsRNA was applied to the roots of A. macrocephala to evaluate its effectiveness in controlling root rot disease. The experimental results showed that F. oxysporum could effectively uptake exogenously applied Tup1-dsRNA, significantly reducing Tup1 gene expression. All lengths of Tup1-dsRNA inhibited fungal growth and caused morphological changes in the fungal hyphae. Further plant experiments and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis indicated that Tup1-dsRNA treatment significantly reduced the incidence of root rot disease in A. macrocephala, which was supported by the reduction in peroxidase (POD) and catalase (CAT) enzyme activities, malondialdehyde (MDA) content, and proline (Pro) levels in treated root tissues. This study demonstrated that exogenous dsRNA could reduce the virulence of F. oxysporum by silencing the Tup1 gene and effectively mitigate the root rot disease it causes in A. macrocephala. The successful application of Tup1-dsRNA provided strong evidence for the potential of RNA interference (RNAi) technology in plant disease control. Future research could further optimize the design and application of dsRNA to enhance its practical value in agriculture. Full article

RNA interference (RNAi) is a regulatory and protective mechanism that plays a crucial role in the growth, development, and control of plant responses to pathogens and abiotic stresses. In spray-induced gene silencing (SIGS), exogenous double-stranded RNAs (dsRNA) are used to efficiently regulate target genes via plant surface treatment. In this study, we aimed to evaluate the effect of specific exogenous dsRNAs on silencing different regions (promoter, protein-coding and intron) of the target SlTRY tomato gene, encoding an R3-type MYB repressor of anthocyanin biosynthesis. We also assessed the impact of targeting different SlTRY regions on the expression of genes involved in anthocyanin and flavonoid biosynthesis. This study demonstrated the critical importance of selecting the appropriate gene target region for dsRNA action. The highest inhibition of the SlTRY gene expression and activation of anthocyanin biosynthesis was achieved by dsRNA complementary to the protein-coding region of SlTRY gene, compared with dsRNAs targeting the SlTRY promoter or intron regions. Silencing the SlTRY gene increased the content of anthocyanins and boosted levels of other substances in the phenylpropanoid pathway, such as caffeoyl putrescine, chlorogenic acid, ferulic acid glucoside, feruloyl quinic acid, and rutin. This study is the first to examine the effects of four different dsRNAs targeting various regions of the SlTRY gene, an important negative regulator of anthocyanin biosynthesis. Full article

The application of double-stranded RNAs (dsRNAs) to plant surfaces has emerged as a promising tool for manipulating gene expression in plants and pathogens, offering new opportunities for crop improvement. While research has shown the capability of exogenous dsRNAs to silence genes, the full spectrum of their impact, particularly on the intricate network of microRNAs (miRNAs), remains largely unexplored. Here, we show that the exogenous application of chalcone synthase (CHS)-encoding dsRNA to the rosette leaves of Arabidopsis thaliana induced extensive alterations in the miRNA profile, while non-specific bacterial neomycin phosphotransferase II (NPTII) dsRNA had a minimal effect. Two days after treatment, we detected 60 differentially expressed miRNAs among the 428 miRNAs found in the A. thaliana genome. A total of 59 miRNAs were significantly changed after AtCHS-dsRNA treatment compared with water and NPTII-dsRNA, and 1 miRNA was significantly changed after AtCHS-dsRNA and NPTII-dsRNA compared with the water control. A comprehensive functional enrichment analysis revealed 17 major GO categories enriched among the genes potentially targeted by the up- and downregulated miRNAs. These categories included processes such as aromatic compound biosynthesis (a pathway directly related to CHS activity), heterocycle biosynthesis, RNA metabolism and biosynthesis, DNA transcription, and plant development. Several predicted targets of upregulated and downregulated miRNAs, including APETALA2, SCL27, SOD1, GRF1, AGO2, PHB, and PHV, were verified by qRT-PCR. The analysis showed a negative correlation between the expression of miRNAs and the expression of their predicted targets. Thus, exogenous plant gene-specific dsRNAs induce substantial changes in the plant miRNA composition, ultimately affecting the expression of a wide range of genes. These findings have profound implications for our understanding of the effects of exogenously induced RNA interference, which can have broader effects beyond targeted mRNA degradation, affecting the expression of other genes through miRNA regulation. Full article

This study aimed to evaluate the effects of arginine (0.5%, 1%, 1.5%, 2%, and 2.5% arginine supplementation levels were selected) on the ovarian development of Pacific white shrimp (Litopenaeus vannamei). The analyzed arginine supplementation levels in each diet were 2.90%, 3.58%, 4.08%, 4.53%, 5.04%, and 5.55%, respectively. A total of 540 shrimp (an initial weight of approximately 14 g) with good vitality were randomly distributed into six treatments, each of which had three tanks (300 L in volume filled with 200 L of water), with 30 shrimp per duplicate. Shrimp were fed three times a day (6:00 a.m., 11:00 a.m., and 6:00 p.m.). The results showed that after the 12-week raring cycle, shrimp fed with 4.08% and 4.53% Arg achieved better ovary development, which was identified by ovarian stage statistics, ovarian morphology observation, serum hormone levels (methylfarneside (MF); 5-hydroxytryptamine (5-HT); estradiol (E2); and gonadotropin-releasing hormone (GnRH)), gene expression (DNA meiotic recombinase 1 (dmc1), proliferating cell nuclear antigen (pcna), drosophila steroid hormone 1 (cyp18a), retinoid X receptor (rxra), and ecdysone receptor (ecr)). Further in-depth analysis showed that 4.08% and 4.53% Arg supplementation increased the concentration of vitellogenin in hepatopancreas and serum (p < 0.05) and upregulated the expression level of hepatopancreatic vg and vgr (p < 0.05), which promoted the synthesis of hepatopancreas exogenous vitellogenin and then transported it into the ovary through the vitellogenin receptor and further promoted ovarian maturation in L. vannamei. Meanwhile, compared with the control group, the expression level of vg in the ovary of the 4.53% Arg group was significantly upregulated (p < 0.05), which indicated endogenous vitellogenin synthesis in ovarian maturation in L. vannamei. Moreover, the expression of genes related to the mechanistic target of the rapamycin complex 1 (mTORC1) pathway and protein levels was regulated by dietary arginine supplementation levels. Arginine metabolism-related products, including nitric oxide synthase (NOS), nitric oxide (NO), and cyclic guanosine monophosphate (cGMP), were also affected. RNA interference was applied here to study the molecular regulation mechanism of arginine on ovarian development in L. vannamei. A green fluorescent protein (GFP)-derived double-stranded RNA (dsGFP) is currently commonly used as a control, while TOR-derived dsRNA (dsTOR) and NOS-derived dsRNA (dsNOS) were designed to build the TOR and NOS in vivo knockdown model. The results showed that the mTORC1 and NO-sGC-cGMP pathways were inhibited, while the vitellogenin receptor and vitellogenin gene expression levels were downregulated significantly in the hepatopancreas and ovary. Overall, dietary arginine supplementation could enhance endogenous and exogenous vitellogenin synthesis to promote ovary development in L. vannamei, and the appropriate dosages were 4.08% and 4.53%. The NO-sGC-cGMP and mTORC1 signaling pathways mediated arginine in the regulation of ovary development in L. vannamei. Full article

The use of double-stranded RNA (dsRNA) for plant protection shows great potential as a sustainable alternative to traditional pesticides. This review summarizes the current state of knowledge on using exogenous dsRNA in plant protection and includes the latest findings on the safety and efficiency of this strategy. The review also emphasizes the need for a cautious and comprehensive approach, considering safety considerations such as off-target effects and formulation challenges. The regulatory landscape in different regions is also discussed, underscoring the need for specific guidelines tailored to dsRNA-based pesticides. The review provides a crucial resource for researchers, regulators, and industry stakeholders, promoting a balanced approach incorporating innovation with thorough safety assessments. The continuous dialog emphasized in this review is essential for shaping the future of dsRNA-based plant protection. As the field advances, collaboration among scientists, regulators, and industry partners will play a vital role in establishing guidelines and ensuring the responsible, effective, and sustainable use of dsRNA in agriculture. Full article