Search Results (23)

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

Dendroctonus armandi (Tsai and Li) (Coleoptera: Curculionidae: Scolytinae) is regarded as the most destructive forest pest in the Qinling and Bashan Mountains of China. The sex determination of Dendroctonus armandi plays a significant role in the reproduction of its population. In recent years, the role of the fem-1 gene in sex determination in other insects has been reported. However, the function and expression of the fem-1 gene in Dendroctonus armandi remain uncertain. In this study, three fem-1 genes were cloned and characterized. These were named Dafem-1A, Dafem-1B, and Dafem-1C, respectively. The expression levels of these three Dafem-1 genes vary at different stages of development and between the sexes. In response to different environmental treatments, including temperature, nutrients, terpenoids, and feeding duration, significant differences were observed between the three Dafem-1 genes at different developmental stages and between males and females. Furthermore, injection of double-stranded RNA (dsRNA) targeting the expressions of the Dafem-1A, Dafem-1B, and Dafem-1C genes resulted in increased mortality, deformity, and decreased emergence rates, as well as an imbalance in the sex ratio. Following the interference with Dafem-1A and Dafem-1C, no notable difference was observed in the expression of the Dafem-1B gene. Similarly, after the interference with the Dafem-1B gene, no significant difference was evident in the expression levels of the Dafem-1A and Dafem-1C genes. However, the interference of either the Dafem-1A or Dafem-1C gene results in the downregulation of the other gene. The aforementioned results demonstrate that the Dafem-1A, Dafem-1B, and Dafem-1C genes play a pivotal role in the regulation of life development and sex determination. Furthermore, it can be concluded that external factors such as temperature, nutrition, terpenoids, and feeding have a significant impact on the expression levels of the Dafem-1A, Dafem-1B, and Dafem-1C genes. This provides a crucial theoretical foundation for further elucidating the sex determination mechanism of Dendroctonus armandi. Full article

Odontotermes formosanus (Shiraki) is a subterranean termite species known for causing severe damage to trees and structures such as dams. During the synergistic evolution of O. formosanus with pathogenic bacteria, the termite has developed a robust innate immunity. Termicin is a crucial antimicrobial peptide in termites, significantly contributing to the defense against external infections. Building upon the successful construction and expression of the dsRNA-HT115 engineering strains of dsOftermicin1 and dsOftermicin2 in our laboratory, this work employs the ultrasonic breaking method to establish an inactivated dsOftermicins-HT115 technological system capable of producing a substantial quantity of dsRNA. This approach also addresses the limitation of transgenic strains which cannot be directly applied. Treatment of O. formosanus with dsOftermicins produced by this method could enhance the virulence of both Bt and Bb to the termites. This study laid the theoretical groundwork for the development of novel termite immunosuppressants and for the advancement and application of termite biological control strategies. Full article

Plant surface treatment with double-stranded RNAs (dsRNAs) has gained recognition as a promising method for inducing gene silencing and combating plant pathogens. However, the regulation of endogenous plant genes by external dsRNAs has not been sufficiently investigated. Also, the effect of the simultaneous application of multiple gene-specific dsRNAs has not been analyzed. The aim of this study was to exogenously target five genes in Arabidopsis thaliana, namely, three transcription factor genes (AtCPC, AtMybL2, AtANAC032), a calmodulin-binding protein gene (AtCBP60g), and an anthocyanidin reductase gene (AtBAN), which are known as negative regulators of anthocyanin accumulation. Exogenous dsRNAs encoding these genes were applied to the leaf surface of A. thaliana either individually or in mixtures. The mRNA levels of the five targets were analyzed using qRT-PCR, and anthocyanin content was evaluated through HPLC-MS. The results demonstrated significant downregulation of all five target genes by the exogenous dsRNAs, resulting in enhanced expression of chalcone synthase (AtCHS) gene and increased anthocyanin content. The simultaneous foliar application of the five dsRNAs proved to be more efficient in activating anthocyanin accumulation compared to the application of individual dsRNAs. These findings hold considerable importance in plant biotechnology and gene function studies. Full article

Bacillus thuringiensis Berliner (Bt) and B. cereus sensu stricto Frankland and Frankland are closely related species of aerobic, spore-forming bacteria included in the B. cereus sensu lato group. This group is one of the most studied, but it remains also the most mysterious species of bacteria. Despite more than a century of research on the features of these ubiquitous bacteria, there are a lot of questionable issues related to their taxonomy, resistance to external influences, endophytic existence, their place in multidimensional relationships in the ecosystem, and many others. The review summarizes current data on the mutualistic relationships of Bt and B. cereus bacteria with plants, the structure of the phytomicrobiomes including Bt and B. cereus, and the abilities of plant-associated and endophytic strains to improve plant resistance to various environmental factors and its productivity. Key findings on the possibility of the use of Cry gene promoter for transcription of the target dsRNA and simultaneous release of pore-forming proteins and provocation of RNA-interference in pest organisms allow us to consider this group of microorganisms as unique tools of genetic engineering and biological control. This will open the prospects for the development and direct change of plant microbiomes, and possibly serve as the basis for the regulation of the entire agroecosystem. Full article

The external application of double-stranded RNA (dsRNA) has recently been developed as a non-transgenic approach for crop protection against pests and pathogens. This novel and emerging approach has come to prominence due to its safety and environmental benefits. It is generally assumed that the mechanism of dsRNA-mediated antivirus RNA silencing is similar to that of natural RNA interference (RNAi)-based defence against RNA-containing viruses. There is, however, no direct evidence to support this idea. Here, we provide data on the high-throughput sequencing (HTS) analysis of small non-coding RNAs (sRNA) as hallmarks of RNAi induced by infection with the RNA-containing potato virus Y (PVY) and also by exogenous application of dsRNA which corresponds to a fragment of the PVY genome. Intriguingly, in contrast to PVY-induced production of discrete 21 and 22 nt sRNA species, the externally administered PVY dsRNA fragment led to generation of a non-canonical pool of sRNAs, which were present as ladders of ~18–30 nt in length; suggestive of an unexpected sRNA biogenesis pathway. Interestingly, these non-canonical sRNAs are unable to move systemically and also do not induce transitive amplification. These findings may have significant implications for further developments in dsRNA-mediated crop protection. Full article

Tumor necrosis is a recurrent characteristic of head and neck squamous cell carcinomas (HNSCCs). There is a need for more investigations on the influence of biomolecules released by these necrotic foci in the HNSCC tumor microenvironment. It is suspected that a fraction of the biomolecules released by necrotic cells are damage-associated molecular patterns (DAMPs), which are known to be natural endogenous ligands of Toll-like receptors (TLRs), including, among others, proteins and nucleic acids. However, there has been no direct demonstration that biomolecules released by HNSCC necrotic cells can activate TLRs. Our aim was to investigate whether some of these molecules could behave as agonists of the TLR3, either in vitro or in vivo. We chose a functional approach based on reporter cell exhibiting artificial TLR3 expression and downstream release of secreted alkaline phosphatase. The production of biomolecules activating TLR3 was first investigated in vitro using three HNSCC cell lines subjected to various pronecrotic stimuli (external irradiation, serum starvation, hypoxia and oxidative stress). TLR3 agonists were also investigated in necrotic tumor fluids from five oral cancer patients and three mouse tumor grafts. The release of biomolecules activating TLR3 was demonstrated for all three HNSCC cell lines. External irradiation was the most consistently efficient stimulus, and corresponding TLR3 agonists were conveyed in extracellular vesicles. TLR3-stimulating activity was detected in the fluids from all five patients and three mouse tumor grafts. In most cases, this activity was greatly reduced by RNAse pretreatment or TLR3 blocking antibodies. Our data indicate that TLR3 agonists are consistently present in necrotic fluids from HNSCC cells and mainly made of dsRNA fragments. These endogenous agonists may induce TLR3, which might lead to a protumorigenic effect. Regarding methodological aspects, our study demonstrates that direct investigations—including functional testing—can be performed on necrotic fluids from patient tumors. Full article

Surface staining has emerged as a rapid technique for applying external stains to trace cellular identities in diverse populations. In this study, we developed a distinctive aptamer with selective binding to cell surface nucleolin (NCL), bypassing cytoplasmic internalization. Conjugation of the aptamer with a FAM group facilitated NCL visualization on live cell surfaces with laser confocal microscopy. To validate the aptamer-NCL interaction, we employed various methods, including the surface plasmon resonance, IHC-based flow cytometry, and electrophoretic mobility shift assay. The G-quadruplex formations created by aptamers were confirmed with a nuclear magnetic resonance and an electrophoretic mobility shift assay utilizing BG4, a G-quadruplex-specific antibody. Furthermore, the aptamer exhibited discriminatory potential in distinguishing between cancerous and normal cells using flow cytometry. Notably, it functioned as a dynamic probe, allowing real-time monitoring of heightened NCL expression triggered by a respiratory syncytial virus (RSV) on normal cell surfaces. This effect was subsequently counteracted with dsRNA transfection and suppressed the NCL expression; thus, emphasizing the dynamic attributes of the probe. These collective findings highlight the robust versatility of our aptamer as a powerful tool for imaging cell surfaces, holding promising implications for cancer cell identification and the detection of RSV infections. Full article

To fulfil the growing needs of the global population, sustainability in food production must be ensured. Insect pests and pathogens are primarily responsible for one-third of food losses and harmful synthetic pesticides have been applied to protect crops from these pests and other pathogens such as viruses and fungi. An alternative pathogen control mechanism that is more “friendly” to the environment can be developed by externally applying double-stranded RNAs (dsRNAs) to suppress gene expression. However, the use of dsRNA sprays in open fields is complicated with respect to variable efficiencies in the dsRNA delivery, and the stability of the dsRNA on and in the plants, and because the mechanisms of gene silencing may differ between plants and between different pathogen targets. Thus, nanocarrier delivery systems have been especially used with the goal of improving the efficacy of dsRNAs. Here, we highlight recent developments in nanoparticle-mediated nanocarriers to deliver dsRNA, including layered double hydroxide, carbon dots, carbon nanotubes, gold nanoparticles, chitosan nanoparticles, silica nanoparticles, liposomes, and cell-penetrating peptides, by review of the literature and patent landscape. The effects of nanoparticle size and surface modification on the dsRNA uptake efficiency in plants are also discussed. Finally, we emphasize the overall limitation of dsRNA sprays, the risks associated, and the potential safety concerns for spraying dsRNAs on crops. Full article

Cuticle coloration in insects is a consequence of the accumulation of pigments in a species-specific pattern. Numerous genes are involved in regulating the underlying processes of melanization and sclerotization, and their manipulation can be used to create externally visible markers of successful gene editing. To clarify the roles for many of these genes and examine their suitability as phenotypic markers in Lygus hesperus Knight (western tarnished plant bug), transcriptomic data were screened for sequences exhibiting homology with the Drosophila melanogaster proteins. Complete open reading frames encoding putative homologs for six genes (aaNAT, black, ebony, pale, tan, and yellow) were identified, with two variants for black. Sequence and phylogenetic analyses supported preliminary annotations as cuticle pigmentation genes. In accord with observable difference in color patterning, expression varied for each gene by developmental stage, adult age, body part, and sex. Knockdown by injection of dsRNA for each gene produced varied effects in adults, ranging from the non-detectable (black 1, yellow), to moderate decreases (pale, tan) and increases (black 2, ebony) in darkness, to extreme melanization (aaNAT). Based solely on its expression profile and highly visible phenotype, aaNAT appears to be the best marker for tracking transgenic L. hesperus. Full article

Recent investigations have shown the possibility of artificial induction of RNA interference (RNAi) via plant foliar treatments with naked double-stranded RNA (dsRNA) to silence essential genes in plant fungal pathogens or to target viral RNAs. Furthermore, several studies have documented the downregulation of plant endogenous genes via external application of naked gene-specific dsRNAs and siRNAs to the plant surfaces. However, there are limited studies on the dsRNA processing and gene silencing mechanisms after external dsRNA application. Such studies would assist in the development of innovative tools for crop improvement and plant functional studies. In this study, we used exogenous gene-specific dsRNA to downregulate the gene of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, in Arabidopsis. The nonspecific NPTII-dsRNA encoding the nonrelated neomycin phosphotransferase II bacterial gene was used to treat plants in order to verify that any observed effects and processing of AtCHS mRNA were sequence specific. Using high-throughput small RNA (sRNA) sequencing, we obtained six sRNA-seq libraries for plants treated with water, AtCHS-dsRNA, or NPTII-dsRNA. After plant foliar treatments, we detected the emergence of a large number of AtCHS- and NPTII-encoding sRNAs, while there were no such sRNAs after control water treatment. Thus, the exogenous AtCHS-dsRNAs were processed into siRNAs and induced RNAi-mediated AtCHS gene silencing. The analysis showed that gene-specific sRNAs mapped to the AtCHS and NPTII genes unevenly with peak read counts at particular positions, involving primarily the sense strand, and documented a gradual decrease in read counts from 17-nt to 30-nt sRNAs. Results of the present study highlight a significant potential of exogenous dsRNAs as a promising strategy to induce RNAi-based downregulation of plant gene targets for plant management and gene functional studies. Full article

Adenosine-to-inosine RNA editing is a system of post-transcriptional modification widely distributed in metazoans which is catalyzed by ADAR enzymes and occurs mostly in double-stranded RNA (dsRNA) before splicing. This type of RNA editing changes the genetic code, as inosine generally pairs with cytosine in contrast to adenosine, and this expectably modulates RNA splicing. We review the interconnections between RNA editing and splicing in the context of human cancer. The editing of transcripts may have various effects on splicing, and resultant alternatively spliced isoforms may be either tumor-suppressive or oncogenic. Dysregulated RNA splicing in cancer often causes the release of excess amounts of dsRNA into cytosol, where specific dsRNA sensors provoke antiviral-like responses, including type I interferon signaling. These responses may arrest cell division, causing apoptosis and, externally, stimulate antitumor immunity. Thus, small-molecule spliceosome inhibitors have been shown to facilitate the antiviral-like signaling and are considered to be potential cancer therapies. In turn, a cytoplasmic isoform of ADAR can deaminate dsRNA in cytosol, thereby decreasing its levels and diminishing antitumor innate immunity. We propose that complete or partial inhibition of ADAR may enhance the proapoptotic and cytotoxic effects of splicing inhibitors and that it may be considered a promising addition to cancer therapies targeting RNA splicing. Full article

The phenomenon of RNA interference (RNAi) is widely used to develop new approaches for crop improvement and plant protection. Recent investigations show that it is possible to downregulate plant transgenes, as more prone sequences to silencing than endogenous genes, by exogenous application of double-stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs). However, there are scarce data on the specificity of exogenous RNAs. In this study, we explored whether plant transgene suppression is sequence-specific to exogenous dsRNAs and whether similar effects can be caused by exogenous DNAs that are known to be perceived by plants and induce certain epigenetic and biochemical changes. We treated transgenic plants of Arabidopsis thaliana bearing the neomycin phosphotransferase II (NPTII) transgene with specific synthetic NPTII-dsRNAs and non-specific dsRNAs, encoding enhanced green fluorescent protein (EGFP), as well as with DNA molecules mimicking the applied RNAs. None of the EGFP-dsRNA doses resulted in a significant decrease in NPTII transgene expression in the NPTII-transgenic plants, while the specific NPTII-dsRNA significantly reduced NPTII expression in a dose-dependent manner. Long DNAs mimicking dsRNAs and short DNA oligonucleotides mimicking siRNAs did not exhibit a significant effect on NPTII transgene expression. Thus, exogenous NPTII-dsRNAs induced a sequence-specific and RNA-specific transgene-suppressing effect, supporting external application of dsRNAs as a promising strategy for plant gene regulation. Full article

The doublesex (dsx) gene, which encodes a transcription factor, regulates sexual differentiation in insects. Sex-specific splicing of dsx occurs to yield male- and female-specific isoforms, which promote male and female development, respectively. Thus, functional disruption of dsx leads to an intersexual phenotype in both sexes. We previously identified a dsx ortholog in the sawfly, Athalia rosae. Similar to dsx in other insects, dsx in the sawfly yields different isoforms in males and females as a result of alternative splicing. The sawfly exploits a haplodiploid mode of reproduction, in which fertilized eggs develop into diploid females, whereas unfertilized eggs parthenogenetically develop into haploid males. In the present study, we knocked down the A. rosae ortholog of dsx (Ardsx) during several developmental stages with repeated double-stranded RNA (dsRNA) injections. Knockdown of Ardsx via parental RNA interference (RNAi), which enables knockdown of genes in offspring embryos, led to a lack of internal and external genitalia in haploid male progeny. Additional injection of dsRNA targeting Ardsx in these animals caused almost complete male-to-female sex reversal, but the resulting eggs were infertile. Notably, the same knockdown approach using diploid males obtained by sib-crossing caused complete male-to-female sex reversal; they were morphologically and behaviorally females. The same RNAi treatment did not affect female differentiation. These results indicate that dsx in the sawfly is essential for male development and its depletion caused complete male-to-female sex reversal. This is the first demonstration of functional depletion of dsx not causing intersexuality but inducing total sex reversal in males instead. Full article

Extracellular vesicles (EVs) carry RNA, proteins, lipids, and diverse biomolecules for intercellular communication. Recent studies have reported that EVs contain double-stranded DNA (dsDNA) and oncogenic mutant DNA. The advantage of EV-derived DNA (EV DNA) over cell-free DNA (cfDNA) is the stability achieved through the encapsulation in the lipid bilayer of EVs, which protects EV DNA from degradation by external factors. The existence of DNA and its stability make EVs a useful source of biomarkers. However, fundamental research on EV DNA remains limited, and many aspects of EV DNA are poorly understood. This review examines the known characteristics of EV DNA, biogenesis of DNA-containing EVs, methylation, and next-generation sequencing (NGS) analysis using EV DNA for biomarker detection. On the basis of this knowledge, this review explores how EV DNA can be incorporated into diagnosis and prognosis in clinical settings, as well as gene transfer of EV DNA and its therapeutic potential. Full article