Search Results (982)

RNA interference (RNAi) represents a promising pest control strategy, applicable to both insect-resistant genetically modified (IRGM) crops and sprayable RNAi insecticides. These products can achieve sequence-specific gene silencing and require rigorous environmental risk assessment (ERA) prior to approval. However, current environmental safety assessments of RNAi products and other RNAi experiments frequently use double-stranded EGFP (dsEGFP) as a negative control, while suitable RNAi-based positive controls are lacking. Sometimes conventional chemical toxins (e.g., chlorpyrifos) or protein inhibitors (e.g., trypsin inhibitors) are used as substitutes, but their distinct mechanisms, persistence, and metabolism make them inappropriate for RNAi-specific evaluations. In this study, we evaluated the suitability of RNAi-based positive controls for assessing non-target effects on Harmonia axyridis, a widely distributed predatory beetle used as a bioindicator in biosafety assessments. Under laboratory conditions, we tested one microRNA (miR-92a) and two double-stranded RNAs (dsHaSnf7 and dsHaDiap1) for their effects on H. axyridis. Injection of miR-92a showed no significant difference in mortality compared to controls, whereas dsHaSnf7 and dsHaDiap1 significantly reduced survival rates and target gene expression, as confirmed by qPCR. These findings suggest that HaSnf7 and HaDiap1 are suitable candidate genes for establishing RNAi-specific positive controls in environmental risk assessments of RNAi-based products. Full article

Fusarium species and the mycotoxins produced by them represent a significant problem for agriculture and human health. Thus, the development of novel management strategies and tools is of high importance. Spray-induced gene silencing (SIGS), based on the natural mechanism of RNA interference (RNAi), has been considered as a highly specific and ecologically safe alternative to chemical fungicides, the use of which is restricted by the emergence of resistant strains and environmental concerns. At the same time, massive application of SIGS is challenged by the degradability of RNA molecules in the environment. Nanoparticles have been widely applied to protect RNA from degradation and improve its action. The aims of this study were to evaluate whether RNAi-mediated silencing of the regulatory FgVe1 gene leads to inhibition of growth, mycotoxin production, and pathogenicity of Fusarium graminearum and whether the use of CaP nanoparticles (CaPs) as double-stranded RNA (dsRNA) carriers enhances and prolongs the silencing effect. It was shown that dsRNA treatment of fungal liquid cultures resulted in 19.78-fold silencing of FgVe1 expression as well as inhibition of expression of genes related to secondary metabolism, including those involved in trichothecene and aurofusarin biosynthesis, thus leading to a reduction in DON accumulation and changes in culture color. The results also demonstrated that naked dsRNA and CaPs:dsRNA nanocomplexes differed in their abilities to induce a high silencing effect at different time points. Naked dsRNA proved more effective in inducing silencing in the early stages of fungal growth, whereas application of nanocomplexes provided a prolonged effect up to 10 days in liquid cultures and up to 14 days on detached leaves. The obtained data can be considered as a basis for the further development of new efficient SIGS-based plant protection strategies. Full article

Management of invasive species is especially difficult when the organisms involved are endophagous and their interactions complex. Such is the case with laurel wilt disease (LWD), a lethal vascular condition caused by Harringtonia lauricola, the fungal symbiont of the non-native redbay ambrosia beetle (RAB), Xyleborus glabratus Eichoff (Coleoptera: Curculionidae). LWD has caused extensive mortality of coastal redbay, Persea borbonia, and is expanding to utilize additional lauraceous hosts, including sassafras, Sassafras albidum. Current management has not been successful in preventing its spread, warranting investigation into additional techniques. RNA interference (RNAi) is a highly specific gene-silencing mechanism used for integrated pest management of crop pests and currently being investigated for use in forests. When targeting essential genes, RNAi can cause rapid insect mortality. Here we focus on RAB, identifying for the first time species-specific reference genes for quantitative real-time PCR (qPCR) and assessing mortality and gene expression after oral ingestion of double-stranded RNAs (dsRNAs) targeting essential genes (hsp, shi, and iap). Our study validates reference genes for expression analyses and shows significant mortality and changes in gene expression for all three target genes. Our research aims to contribute to the development of innovative management strategies for this invasive pest complex. Full article

Amphioxus belongs to the subphylum Cephalochordata and occupies a transitional position in evolution between invertebrates and vertebrates. Due to the lack of viruses suitable for immunostimulation in amphioxus, this study for the first time explored the pathogenicity and waterborne transmission of Grass Carp Reovirus (GCRV), a double-stranded RNA virus, during its infection of amphioxus. Soaking amphioxus in GCRV suspension can cause obvious damage to gill tissues and severely disrupt the structure of gill filaments. The virus survived in seawater for no more than 48 h. Infection kinetics studies showed that the expression of VP5 (a viral capsid protein) mRNA in gill tissues peaked at 14 h. After co-culturing GCRV-infected amphioxus with healthy amphioxus for 72 h, the gills of healthy amphioxus showed obvious pathological damage. Additionally, the presence of the virus was verified by RT-PCR amplification of VP5 expression, indicating that GCRV can be transmitted via water. Transcriptome sequencing analysis showed that the Mitogen-Activated Protein Kinase (MAPK), calcium signaling pathway, and chitin metabolic pathway were significantly activated in amphioxus after GCRV stimulation. This study confirmed that GCRV can infect cephalochordates, revealing its gill-tropism and water-borne transmission ability, providing a new perspective for studying the cross-species infection mechanism of aquatic viruses and the prevention and control of aquatic diseases. Full article

Allergenic proteins in natural rubber latex (NRL) pose significant health risks, particularly in rubber gloves. This study evaluated RNA interference (RNAi) technology for silencing HbREF (rubber elongation factor) and HbSRPP (small rubber particle protein) genes in Hevea brasiliensis to reduce latex allergen content. Double-stranded RNA (dsRNA) targeting these genes demonstrated high stability at 25–37 °C for 6 h and under UV/outdoor conditions for 72 h, but degraded rapidly above 50 °C. Among the three delivery methods tested, direct injection achieved the highest efficiency (>90% gene silencing within 12 h), followed by root drenching (54–84%) and foliar spray (46–70%). HbREF silencing achieved 98–99% expression reduction within 3 h, while HbSRPP showed dose-dependent responses (70–90% silencing) without off-target effects. Gene silencing affected downstream rubber synthesis genes HbCPT (cis-prenyltransferase) and HbRME (rubber membrane elongation protein) (37–58% reduction) while upstream genes remained unaffected. HbREF silencing reduced Hev b1 allergen by 64.04% and Hev b3 by 12.51%, whereas HbSRPP silencing decreased Hev b3 by 71.54% and Hev b1 by 13.48%. Both treatments caused only a 11–13% reduction in dry rubber content. This RNAi approach effectively reduces major latex allergens while maintaining rubber production, demonstrating commercial potential for developing hypoallergenic rubber products through precision agriculture biotechnology. Full article

The nourishment of the human population depends on a handful of staple crops, such as maize, rice, wheat, soybeans, potatoes, tomatoes, and cassava. However, all crop plants are affected by at least one virus causing diseases that reduce yield, and in some parts of the world, this leads to food insecurity. Conventional management practices need to be improved to incorporate recent scientific and technological developments such as antiviral gene silencing, the use of double-stranded RNA (dsRNA) to activate an antiviral response, and nanobiotechnology. dsRNA with antiviral activity disrupt viral replication, limit infection, and its use represents a promising option for virus management. However, currently, the biggest limitation for viral diseases management is that dsRNA is unstable in the environment. This review is focused on the potential of nanoparticles and nanocarriers to deliver dsRNA, enhance stability, and activate antiviral gene silencing. Effective carriers include metal-based nanoparticles, including silver, zinc oxide, and copper oxide. The stability of dsRNA and the efficiency of gene-silencing activation are enhanced by nanocarriers, including layered double hydroxides, chitosan, and carbon nanotubes, which protect and transport dsRNA to plant cells. The integration of nanocarriers and gene silencing represents a sustainable, precise, and scalable option for the management of viral diseases in crops. It is essential to continue interdisciplinary research to optimize delivery systems and ensure biosafety in large-scale agricultural applications. Full article

As one of the key vectors for the transmission of Dengue fever, Aedes albopictus is highly ecologically adaptable. The development of environmentally compatible biological defence and control technologies has therefore become an urgent need for vector biological control worldwide. This study constructed and used double-stranded RNA (dsRNA) expression vectors targeting the cyp314a1 and cyp315a1 genes of Ae. albopictus to transform Chlamydomonas reinhardtii and Chlorella vulgaris, achieving RNA interference (RNAi)-mediated gene silencing. The efficacy of the RNAi recombinant algal strain biocide against Ae. albopictus was evaluated by administering it to Ae. albopictus larvae. The results showed that the oral administration of the cyp314a1 and cyp315a1 RNAi recombinant C. reinhardtii/C. vulgaris strains was lethal to Ae. albopictus larvae and severely affected their pupation and emergence. The recombinant algal strains triggered a burst of ROS (Reactive Oxygen Species) in the mosquitoes’ bodies, resulting in significant increases in the activities of the superoxide dismutase (SOD), peroxiredoxin (POD) and catalase (CAT), as well as significant upregulation of the mRNA levels of the CME pathway genes in larvae. In the simulated field experiment, the number of Ae. albopictus was reduced from 1000 to 0 in 16 weeks by the RNAi recombinant Chlorella, which effectively controlled the population of mosquitoes. Meanwhile, the levels of nitrogen (N), phosphorus (P), nitrate, nitrite, ammonia and COD (Chemical Oxygen Demand) in the test water decreased significantly. High-throughput sequencing analyses of 18S rDNA and 16S rDNA showed that, with the release of RNAi recombinant Chlorella into the test water, the biotic community restructuring dominated by resource competition caused by algal bloom, as well as the proliferation of anaerobic bacteria and the decline of aerobic bacteria triggered by anaerobic conditions, are the main trends in the changes in the test water. This study is an important addition to the use of RNAi recombinant microalgae as a biocide. Full article

Pediatric tumors such as neuroblastoma are characterized by a genome-wide ‘transcriptional burden’, surmising the involvement of multiple alterations of gene expression. Search for master regulators of transcription whose inactivation is lethal for tumor cells identified the non-POU domain-containing octamer-binding protein (NONO), a member of the Drosophila Behavior/Human Splicing family known for the ability to form complexes with macromolecules. NONO emerges as an essential mechanism in normal neurogenesis as well as in tumor biology. In particular, NONO interactions with RNAs, largely with long non-coding MYCN transcripts, have been attributed to the aggressiveness of neuroblastoma. Broadening its significance beyond MYCN regulation, NONO guards a subset of transcription factors that comprise a core regulatory circuit, a self-sustained loop that maintains transcription. As a component of protein–protein complexes, NONO has been implicated in the control of cell cycle progression, double-strand DNA repair, and, generally, in cell survival. Altogether, the pro-oncogenic roles of NONO justify the need for its inactivation as a therapeutic strategy. However, considering NONO as a therapeutic target, its druggability is a challenge. Recent advances in the inactivation of NONO and downstream signaling with small molecular weight compounds make promising the development of pharmacological antagonists of NONO pathway(s) for neuroblastoma treatment. 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 14-3-3 protein family, which includes the isoforms η, γ, ε, θ, β, and ζ, is essential for controlling a number of pathways linked to DNA and RNA viruses, including HIV, influenza A virus (IAV), measles virus, HRSV, and double-stranded DNA viruses. TRIM32, an E3 ubiquitin ligase, has been reported to target IAV’s PB1 polymerase for species-specific degradation via ubiquitination. Notably, 14-3-3η binds to phosphorylated TRIM32, preventing its autoubiquitylation and forming soluble but inactive cytoplasmic aggregates that regulate TRIM32 levels. However, the functional link between 14-3-3η, TRIM32, and PB1 during viral infection remains unclear. In this study, we establish a mechanistic connection between 14-3-3η–TRIM32 and TRIM32–PB1 interactions in IAV (H1N1) infection. We demonstrate that 14-3-3η directly interacts with PB1, influencing viral replication. Using transient knockdown models, we show that 14-3-3η deficiency alters influenza virus-induced cytotoxicity, cell death, immune responses, and reactive oxygen species (ROS) production. Additionally, we observe a significant reduction in the soluble TRIM32 levels in 14-3-3η-deficient cells, which leads to increased PB1 accumulation and thus suggests a critical regulatory role for 14-3-3η in PB1 stability. Our findings reveal a novel function of 14-3-3η in influenza virus infection, demonstrating its role in PB1 regulation via TRIM32 and its impact on innate immune activation. This study highlights 14-3-3η as a possible target for antiviral treatments against influenza and offers fresh insights into the host–virus relationship. Full article

Mitochondria are crucial for a wide range of cellular processes. One of the most important is innate immunity regulation. Apart from functioning as a signaling hub in immune reactions, mitochondrial nucleic acids can themselves act as damage-associated molecular patterns (DAMPs) to participate in immune processes directly. This review synthesizes the current understanding of mitochondrial RNA (mtRNA) biology and its link to immune activation through aberrant accumulation. We focus on its origin through bidirectional mitochondrial transcription and metabolism, encompassing maturation (cleavage, polyadenylation, modification) and degradation. Dysregulation of mtRNA metabolism leads to mt-dsRNA (mitochondrial double-stranded RNA) accumulation, which escapes mitochondria via specific channels into the cytosol and serves as DAMPs to trigger an immune response. We discuss the critical roles of key regulatory factors, including PNPT1 (PNPase, Polyribonucleotide Nucleotidyltrans ferase 1), in controlling mt-dsRNA levels and preventing inappropriate immune activation. Finally, we review the implications of mt-dsRNA-driven inflammation in human diseases, including autoimmune disorders, cellular senescence, and viral infection pathologies, highlighting unresolved questions regarding mt-dsRNA release mechanisms. Full article

CRISPR/Cas9 gene editing technology is widely used in plant gene editing to verify gene function or improve agronomic traits. In the CRISPR/Cas9 system, Cas9 expression hinges on promoter choice, and CRISPR/Cas9 driven by a strong promoter or cell division-specific promoter has a higher editing efficiency. The CRISPR/Cas9 mechanism involves the CAS9 enzyme, which, directed by guide RNA, cleaves target double-stranded DNA and subsequently induces insertions or deletions (InDels) through the non-homologous end joining (NHEJ) repair pathway. The Ku protein plays a central role in the NHEJ repair process. It remains unclear whether driving Cas9 with promoters of AtKu70 and AtKu80, which are subunits of the Ku protein, will enhance gene editing efficiency. In this study, the promoters of AtKu70 and AtKu80 were cloned and used to drive Cas9 in the CRISPR/Cas9 system. Four different genes, GmRj7, GmNNL1, AtPDS3, and AtBRI1, were designed for soybean hairy root transformation and Arabidopsis transformation. The results showed that the CRISPR/Cas9 systems driven by the promoters of AtKu70 and AtKu80 achieved higher homozygous/biallelic mutation efficiencies than the CRISPR/Cas9 system driven by the 35S promoter in hairy root transformation by Rhizobium rhizogenes and stable genetic transformation with Rhizobium tumefaciens. Full article

Cyrtomium hemionitis is a Cyrtomium fern with potential medicinal value; however, the lack of chloroplast genome data for this species limits its utilization and exploitation. In this study, the Illumina NovoSeq 6000 platform and SPAdes v3.14.1 were used to sequence and assemble the chloroplast genome of C. hemionitis. The chloroplast genome was 151,295 bp in length and exhibited a typical circular, double-stranded, quadripartite plastome architecture, with a GC content of 42.43%. Additionally, it included 30 high-frequency codons, 26 of which ended with A or U. In total, we annotated 130 coding genes, which included 88 protein-coding genes, 8 rRNA genes, and 34 tRNA genes. The IR (inverted repeat) boundaries of the genus Cyrtomium differed from those of common plants, with differences discovered in the JLB (large single-copy, inverted repeat b) and JLA (large single-copy, inverted repeat a) boundaries in this genus. Additionally, the phylogeny of this genus showed that C. hemionitis was more closely related to C. falcatum, whereas Dryopteris crassirhizoma was closely related to the genus Cyrtomium. These findings have significant implications for future research and can serve as a reference for the molecular evolution, systematic development, and utilization of C. hemionitis. Full article

The dengue virus (DENV) exploits host cell exosome pathways to disseminate and evade immunity. However, the host factors enabling this process remain poorly defined. Here, we demonstrate that DENV infection robustly induces expression of the short isoform of Reticulon 3 (RTN3S) in hepatic (Huh7) and monocytic cells, and that RTN3S is a critical driver of infectious exosome biogenesis. RTN3S physically associates with double-stranded viral RNA and the DENV non-structural protein 3 (NS3) in infected cells, indicating its integration into the viral replication complex. Loss of RTN3 markedly reduced exosome production and the exosomal export of viral RNA and proteins, demonstrating that RTN3S is required for efficient exosome-mediated viral release. Conversely, overexpression of full-length RTN3S dramatically increased the release of infectious virus-containing exosomes; truncation of the RTN3S C-terminal domain abolished this enhancement, confirming the essential role of the C-terminus in RTN3S’s pro-viral exosomal function. In DENV-infected monocytes, we observed a shift toward a CD16-positive intermediate phenotype, accompanied by the upregulation of genes involved in vesicle biogenesis and stress response. These infected monocytes also secreted higher levels of inflammatory cytokines. Similarly, monocytes from Dengue patients exhibited high RTN3 expression, which correlated with an expansion of intermediate (CD16⁺) subsets and enriched expression of vesicle trafficking machinery genes. These findings reveal a previously unrecognized mechanism by which DENV hijacks RTN3S to promote the formation of infectious exosomes, thereby facilitating viral dissemination and immune evasion. RTN3S thus represents a novel element of the Dengue pathogenesis and a potential target for host-directed antiviral strategies. Full article

Macrophomina phaseolina is a widely distributed soilborne phytopathogenic fungus that causes destructive diseases such as charcoal rot and stem canker, posing serious threats to crop yield and quality. In recent years, mycoviruses have gained attention as potential biological control agents. In this study, a novel double-stranded RNA (dsRNA) virus was identified from M. phaseolina strain 22C-8, isolated from sesame (Sesamum indicum L.) charcoal rot samples in Fuyang, Anhui Province, China. The viral genome comprised four dsRNA segments, each encoding a single open reading frame (ORF) predicted to encode RNA-dependent RNA polymerase (RdRp), coat protein (CP), and two hypothetical proteins. Phylogenetic analysis classified the virus as a new member of the genus Betachrysovirus in the family Chrysoviridae, and it was designated Macrophomina phaseolina chrysovirus 2 (MpChrV2). Pathogenicity assays in sesame seedlings revealed that MpChrV2 infection significantly reduced the virulence of M. phaseolina strain 22C-8. In contrast, virus-free derivatives (22C-8-VF18), obtained via protoplast regeneration, caused more severe symptoms and exhibited enhanced growth rates, indicating that MpChrV2 alters fungal physiology and pathogenicity. These findings suggest that MpChrV2 possesses a typical hypovirulence phenotype and holds promise as a biocontrol agent for sesame charcoal rot. Full article