Search Results (100)

Small auxin-up-regulated RNA (SAUR) genes are involved in the regulation of dynamic and adaptive growth in higher plants. However, their function and mode of action in citrus root growth are still unknown. Here, we demonstrate that in Poncirus trifoliata, PtrSAUR32 acted downstream of the auxin response factor PtrARF8 to regulate root growth by interacting with PtrPP2C.Ds, subfamily type 2C protein phosphatases which interacted with H-ATPase and PtrHA. In this study, several members of SAUR family in Poncirus trifoliata are identified to be associated with the growth and development of the roots. Among them, PtrSAUR32 was found to be highly expressed in the RT (root tip), and the level of its expression was significantly positively corelated to the length of primary roots (p < 0.01). The overexpression of PtrSAUR32 in citrus significantly promoted the growth of primary roots. In PtrSAUR32 transgenic citrus plants, the expressions of several auxin biosynthesis and transport genes were altered in accordance with the expression of PtrSAUR32. Y1H and dual-luciferase reporter assays proved that the expression of PtrSUAR32 is regulated by PtrARF8. Y2H and BiFC assay results indicated that PtrSAUR32 interacted with PtrPP2C.Ds subfamily members PtrPP2C.D1, PtrPP2C.D6, and PtrPP2C.D7, of which PtrPP2C.D7 could interact with PtrHA in vivo. Full article

The Colorado Potato Beetle (CPB, Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) remains a destructive agricultural pest worldwide that continually overcomes conventional control methods. In recent years, RNA interference (RNAi) has emerged as an alternative for its management; however, although promising results have been reported, its effectiveness has been influenced by several factors, including the length of double-stranded RNA (dsRNA), the delivery method, stability, and especially the selection of the target gene. In this study, we designed and synthesized 290 bp dsRNAs targeting the SERCA and CPR genes from L. decemlineata, which encode the Sarco/Endoplasmic Reticulum Ca²⁺-ATPase and NADPH–Cytochrome P450 Reductase, respectively. Both dsRNAs successfully reduced transcript levels in larvae, with dsSERCA achieving ~60% knockdown by day 3 and dsCPR achieving ~50% knockdown by day 7. Furthermore, both treatments affected the larval growth and survival rate. However, while the dsCPR-treated larvae showed a 59% reduction in weight gain, the administration of dsSERCA had a strong phenotypic effect on the larvae, leading to decreased feeding, a 50.4% reduction in weight gain, and ultimately, 100% mortality. These results suggest that the SERCA and CPR genes could be promising targets for L. decemlineata control and emphasize the importance of appropriate target gene selection for RNAi silencing, as well as the need to explore and validate new genes for RNAi-mediated pest management. Full article

Mesothelioma is a rare and aggressive cancer linked to asbestos exposure and characterized by rapid metastasis and poor prognosis. Inhibition of adenosine deaminase acting on dsRNA 2 (ADAR2) RNA binding but not ADAR2 editing has shown antitumor effects in mesothelioma. Natural compounds from the Traditional Chinese Medicine (TCM) database were docked to the RNA-binding interface of ADAR2’s second dsRNA binding domain (dsRBD2), and their drug-likeness and predicted safety were assessed. Eight ligands (ZINC000085597263, ZINC000085633079, ZINC000014649947, ZINC000034512861, ZINC000070454124, ZINC000085594944, ZINC000085633008, and ZINC000095909822) showed high binding affinity to dsRBD2 from molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations. Protein–ligand interactions were analyzed to identify key residues contributing to these binding affinities. Molecular dynamics (MD) simulations of dsRBD–ligand–RNA complexes revealed that four compounds (ZINC000085597263, ZINC000085633079, ZINC000014649947, and ZINC000034512861) had negative binding affinities to dsRBD2 in the presence of the RNA substrate GluR-2. Key residues, including Val164, Met165, Lys209, and Lys212, were crucial for ligand binding, even with RNA present, suggesting these compounds could inhibit dsRBD2’s RNA-binding function. The predicted biological activities of these compounds indicate potential anticancer properties, particularly for the treatment of mesothelioma. These compounds are structurally similar to known anti-mesothelioma agents or anticancer drugs, highlighting their therapeutic potential. Current mesothelioma treatments are limited. Optimization of these compounds, alone or in combination with current therapeutics, has potential for mesothelioma treatment. Additionally, five high-quality full-length ADAR2 models were developed. These models provide insights into ADAR2 function, mutation impacts, and potential areas for protein engineering to enhance stability, RNA-binding specificity, or protein interactions, particularly concerning dimerization or complex formation with other proteins and RNAs. Full article

Cathepsin B is a member of the cysteine protease family and plays an important role in the innate immunity of aquatic invertebrates. A previous study identified that Cathepsin B-like (CTSB-l) may be involved in the response of alkali treatment in Macrobrachium nipponense. The present study aims to identify the potential regulatory roles of CTSB-l in the response of alkali treatment in M. nipponense through performing the quantitative real-time PCR analysis (qPCR), in situ hybridization (ISH) analysis, and RNA interference (RNAi) analysis. The full length of the MnCTSB-l cDNA was 1272 bp with an open reading frame of 987 bp, encoding 328 amino acids. Phylogenetic tree analysis indicated that the amino acid sequence of MnCTSB-l is highly homologous to those of crustacean cathepsin B-like. qPCR analysis showed that MnCTSB-l mRNA is expressed in all tested tissues with the highest level of expression in hepatopancreas in both male and female prawns. The expressions of MnCTSB-l were significantly stimulated in gills under the alkali concentration of both 5 mmol/L and 10 mmol/L, predicting that this gene may be involved in the response of alkali treatment in M. nipponense, which was consistent with the previous study. ISH showed that MnCTSB-l signals were mainly observed in the hemolymph vessels and membranes of gills, as well as in the basement membranes of hepatopancreas, in both male and female prawns. RNAi analysis revealed that the injection of double-stranded RNA of CTSB (dsCTSB) resulted in a significant decrease in MnCTSB-l expressions. In addition, prawn cumulative mortality was significantly higher in the dsCTSB-injected group, compared to that of dsGFP-injected group, under alkali treatments of both 5 mmol/L and 10 mmol/L, indicating CTSB-l plays an essential role in regulating alkalinity acclimation in M. nipponense. The present study identifies the regulatory functions of CTSB-l in the response of alkali treatment in M. nipponense, promoting the survival rate and aquaculture of this species in a water environment with high alkalinity. Full article

Double-stranded RNA (dsRNA) biopesticides offer the potential for highly targeted pest control with minimal off-target impacts. Varroa mites (Varroa destructor) are an important pest of honey bees (Apis mellifera) that are primarily managed by synthetic pesticides. A next-generation treatment using a varroa-active dsRNA, vadescana, has been developed to target calmodulin expression in varroa. We evaluated the potential exposure of non-target species to vadescana. First, we assessed potential gene silencing effects on 39 arthropods with known genomes via bioinformatics. Three mite species, monarch butterflies (Danaus plexippus), fruit flies (Drosophila melanogaster), and European earwigs (Forficula auricularia) showed theoretical potential for off-target effects. These in silico results could be used to help inform risk assessments. Second, we conducted vadescana feeding trials on the greater wax moth (Galleria mellonella), a common beehive associate. There were no significant differences in wax moth reproduction, survival, or adult F2 wing length between vadescana-fed and control groups. Male F2 body weight was slightly but significantly lower in wax moths exposed to the highest vadescana dose, with no such effect observed in female moths. Calmodulin gene expression was unaffected in wax moths. Our hazard assessment of vadescana’s lethal and sublethal effects on wax moths indicates minimal impact following continuous dietary exposure far greater than any exposure that might be expected in the field, in line with the bioinformatics findings. This biopesticide appears highly varroa-specific and likely has fewer non-target effects than many current varroa control methods. Full article

Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bur) has a circular single-stranded ssDNA genome of 2759 nucleotides in length and belongs to the genus Begomovirus (family, Geminiviridae). CLCuKoV-Bur causes cotton leaf curl disease (CLCuD) and is transmitted by the whitefly Bemisis tabaci cryptic species. Monopartite begomoviruses encode five open reading frames (ORFs). CLCuKoV-Bur replicates through a dsDNA intermediate. Five open reading frames (ORFs) are organized in the small circular, single-stranded (ss)-DNA genome of CLCuKoV-Bur (2759 bases). RNA interference (RNAi) is a naturally occurring process that has revolutionized the targeting of gene regulation in eukaryotic organisms to combat virus infection. The aim of this study was to elucidate the potential binding attractions of cotton-genome-encoded microRNAs (Gossypium hirsutum-microRNAs, ghr-miRNAs) on CLCuKoV-Bur ssDNA-encoded mRNAs using online bioinformatics target prediction tools, RNA22, psRNATarget, RNAhybrid, and TAPIR. Using this suite of robust algorithms, the predicted repertoire of the cotton microRNA-binding landscape was determined for a CLCuKoV-Bur consensus genome sequence. Previously experimentally validated cotton (Gossypium hirsutum L.) miRNAs (n = 80) were selected from a public repository miRNA registry miRBase (v22) and hybridized in silico into the CLCuKoV-Bur genome (AM421522) coding and non-coding sequences. Of the 80 ghr-miRNAs interrogated, 18 ghr-miRNAs were identified by two to four algorithms evaluated. Among them, the ghr-miR399d (accession no. MIMAT0014350), located at coordinate 1747 in the CLCuKoV-Bur genome, was predicted by a consensus or “union” of all four algorithms and represents an optimal target for designing an artificial microRNA (amiRNA) silencing construct for in planta expression. Based on all robust predictions, an in silico ghr-miRNA-regulatory network was developed for CLCuKoV-Bur ORFs using Circos software version 0.6. These results represent the first predictions of ghr-miRNAs with the therapeutic potential for developing CLCuD resistance in upland cotton plants. Full article

Banana streak GF virus (BSGFV) is the extremely dangerous monopartite badnavirus (genus, Badnavirus; family, Caulimoviridae) of banana (Musa acuminata AAA Group) that imposes a serious threat to global banana production. The BSGFV causes a devastating pandemic in banana crops, transmitted by deadly insect pest mealybug vectors and replicated through an RNA intermediate. The BSGFV is a reverse-transcribing DNA virus that has a monopartite open circular double-stranded DNA (dsDNA) genome with a length of 7325 bp. RNA interference (RNAi) is a natural mechanism that has revolutionized the target gene regulation of various organisms to combat virus infection. The current study aims to locate the potential target binding sites of banana-encoded microRNAs (mac-miRNAs) on the BSGFV-dsDNA-encoded mRNAs based on three algorithms, RNA22, RNAhybrid and TAPIR. Mature banana (2n = 3x = 33) miRNAs (n = 32) were selected and hybridized to the BSGFV genome (MN296502). Among the 32 targeted mature locus-derived mac-miRNAs investigated, two banana mac-miRNA homologs (mac-miR162a and mac-miR172b) were identified as promising naturally occurring biomolecules to have binding affinity at nucleotide positions 5502 and 9 of the BSGFV genome. The in silico banana-genome-encoded mac-miRNA/mbg-miRNA-regulatory network was developed with the BSGFV—ORFs using Circos software (version 0.69-9) to identify potential therapeutic target proteins. Therefore, the current work provides useful biological material and opens a new range of opportunities for generating BSGFV-resistant banana plants through the genetic manipulation of the selected miRNAs. Full article

In the search for effective strategies to control the Colorado Potato Beetle, RNA interference technology has emerged as a promising method due to its capacity to suppress genes selectively. Factors such as the target gene and double-stranded RNA (dsRNA) length are critical for optimizing gene silencing efficiency. In this study, we designed and synthesized in vitro dsRNAs of varying lengths targeting the ACE1 gene, which encodes the AChE1 isoform of acetylcholinesterase in the beetle. All tested dsRNA lengths (222 bp, 543 bp, 670 bp, and 870 bp) promoted transcript reduction. The 670 bp dsRNA was the most effective, reducing transcript levels by approximately 40% by day seven, followed by the 543 bp dsRNA. No significant differences were observed between the 222 bp and 870 bp dsRNAs. Furthermore, all of the dsRNA lengths resulted in reduced weight gain and increased mortality in larvae, with the 670 bp dsRNA showing the highest mortality rate, leaving only 63% larval survival, a trend that persisted through day nine. These findings emphasize that dsRNA length is a key factor in the silencing response, underscoring the importance of selecting the optimal length while considering the gene’s target, stability, and delivery methods. This study contributes to establishing design criteria for dsRNA, aiding in the development of more effective and sustainable pest management strategies. Full article

Salmonella is one of the main foodborne pathogens. Irrational antibiotic management has led to an increase in the incidence of multidrug-resistant strains. Bacteriophages may be an alternative method of food biopreservation and contribute to reducing the number of food poisonings requiring pharmacotherapy. This study aimed to isolate a bacteriophage (phage) targeting indigenous multidrug-resistant (MDR) Salmonella strains, followed by their biological, morphological, and genomic characterization. In this study we isolated Salmonella phage KKP_3822, targeting MDR Salmonella Manchester strain KKP 1213. Salmonella phage KKP_3822 retained high activity in the temperature range from −20 °C to 40 °C and active acidity from pH 3 to 11. Temperatures of 70 °C and 80 °C and extreme pH values (2 and 12) significantly reduced the phage titer. Its activity decreased proportionally to the time of UV exposure. Genome analysis (linear dsDNA with a length of 114,843 bp) revealed the presence of 27 tRNA genes. Proteins encoded by the vB_Sen-IAFB3822 phage were divided into functional modules related to (i) phage structure/assembly, (ii) DNA replication/modification/regulation, (iii) phage lysis, and (iv) DNA packaging into the capsid. No genes associated with antibiotic resistance or integration into the host genome, markers of temperate bacteriophages, were annotated in the Salmonella phage KKP_3822 genome. Based on morphological features and whole-genome sequence analysis, the newly isolated Salmonella phage KKP_3822 shows the greatest similarity to representatives of tailed phages from the Caudoviricetes class, Demerecviridae family, and Epseptimavirus genus. Genome analysis confirmed the virulent nature of the Salmonella phage KKP_3822, making it a potential candidate for food biocontrol. Full article

Background/Objectives: Antimicrobial resistance (AMR) is a major public health threat, which is exacerbated by the lack of new antibiotics and the emergence of multidrug-resistant (MDR) superbugs. Comprehensive efforts and alternative strategies to combat AMR are urgently needed to prevent social, medical, and economic consequences. Pseudomonas aeruginosa is a pathogen responsible for a wide range of infections, from soft tissue infections to life-threatening conditions such as bacteremia and pneumonia. Bacteriophages have been considered as a potential therapeutic option to treat bacterial infections. Our aim was to isolate phages able to infect MDR P. aeruginosa strains. Methods: We isolated two lytic phages, using the conventional double layer agar technique (DLA), from samples obtained from the influent of a wastewater treatment plant in Concepción, Chile. The phages, designated as PaCCP1 and PaCCP2, were observed by electron microscopy and their host range was determined against multiple P. aeruginosa strains using DLA. Moreover, their genomes were sequenced and analyzed. Results: Phage PaCCP1 is a member of the Septimatrevirus genus and phage PaCCP2 is a member of the Pbunavirus genus. Both phages are tailed and contain dsDNA. The genome of PaCCP1 is 43,176 bp in length with a GC content of 54.4%, encoding 59 ORFs, one of them being a tRNA gene. The genome of PaCCP2 is 66,333 bp in length with a GC content of 55.6%, encoding 102 non-tRNA ORFs. PaCCP1 is capable of infecting five strains of P. aeruginosa, whereas phage PaCCP2 is capable of infecting three strains of P. aeruginosa. Both phages do not contain bacterial virulence or AMR genes and contain three and six putative Anti-CRISPR proteins. Conclusions: Phages PaCCP1 and PaCCP2 show promise as effective treatments for MDR P. aeruginosa strains, offering a potential strategy for controlling this clinically important pathogen through phage therapy. Full article

Mikania micrantha (“mile-a-minute” weed) is a global invasive alien weed that can cause severe damage to agroforestry ecosystems and significant agricultural losses worldwide. Although chemical, manual, or mechanical control methods are widely used to control M. micrantha, RNA interference (RNAi)-based biocontrol methods have rarely been reported for this species. The MONOPTEROS (MP) gene, encoding an auxin response factor, plays an essential role in embryonic root initiation in Arabidopsis thaliana. In this study, we identified the MP gene from M. micrantha via orthologous gene analysis. A total of 37 MP orthologous genes was identified in 4 plants, including 9 MP candidate genes in M. micrantha, 13 in Helianthus annuus, 6 in Chrysanthemum nankingense, and 9 in Lactuca sativa. Phylogenetic analysis revealed that an MP candidate gene in M. micrantha (Mm01G000655, named MmMP) was clustered into one clade with the MP gene in A. thaliana (AtMP). In addition, both MmMP and AtMP contain a B3-DNA binding domain that is shared by transcription factors that regulate plant embryogenesis. To study gene function, dsRNA against MmMP (dsMmMP) was applied to the roots of M. micrantha. Compared with those of the controls, the expression of MmMP was reduced by 43.3%, 22.1%, and 26.2% on the first, third, and fifth days after dsMmMP treatment, respectively. The dsMmMP-treated plants presented several morphological defects, mostly in the roots. Compared with water-treated plants, the dsMmMP-treated plants presented reduced developmental parameters, including root length, number of adventitious roots, root fresh and dry weights, plant height, and aboveground biomass. Additionally, safety assessment suggested that this dsMmMP treatment did not silence MP genes from non-target plants, including rice and tomato; nor did it inhibit root growth in those species. Collectively, these results suggest that MmMP plays an important role in root development in M. micrantha and provides a potential target for the development of species-specific RNAi-based herbicides. Full article

In this study, we investigated the regulatory roles of the juvenile hormone epoxide hydrolase (JHEH) gene in the reproductive process of female Macrobrachium nipponense. Its total cDNA length was 1848 bp, encoding for 460 amino acids. It contained conserved domains typical of epoxide hydrolases, such as the Abhydrolase family domain, the EHN epoxide hydrolase superfamily domain, and the “WWG” and “HGWP” motifs. The qPCR results showed that the expression of Mn-JHEH was the highest in hepatopancreas. Mn-JHEH was expressed at all stages of the embryonic and larval stages. The expression of Mn-JHEH at different developmental periods of the ovary was positively correlated with ovarian maturation. In situ hybridization showed that it was mainly located in the cytoplasmic membrane and nucleus of oocytes. The RNA interference technique was used to study the role of Mn-JHEH in the process of ovarian maturation. The knockdown of Mn-JHEH with dsRNA in the experimental group resulted in a significant decrease in the percentage of ovaries exceeding stage O-III and the gonadal index compared with the control group. On day 14 (the second molt), the molt frequency was significantly higher in the control group than in the experimental group. The results showed that Mn-JHEH played an important role in ovarian maturation and molting. 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

Cardiomyopathy syndrome (CMS) caused by piscine myocarditis virus (PMCV) has emerged with the rise of the aquaculture of Atlantic salmon (Salmo salar). The lack of cell culture cultivation has hampered the study of this infection. In this study, samples from naturally PMCV-infected Atlantic salmon from different commercial farms were collected and used. In situ hybridization (ISH) revealed intense staining of PMCV RNA in myocardial cells in the spongiform layer of the heart ventricle but almost no staining in the compact layer. In the kidneys, only sporadic staining was seen. Viral RNA was present in all organs, with the highest loads in the heart, kidney, and spleen. The high viral PMCV RNA loads in the heart were due to extensive viral mRNA transcription. The high ratio of viral mRNA to viral genomic dsRNA indicated active transcription but limited production of new viral particles. This suggests that the histopathological changes in the heart are caused by viral mRNA and corresponding viral proteins and not by virus particle formation. The production of full-length transcripts is regulated, with a reduction in the relative number of ORF3-containing transcripts at high transcription rates. Efforts to identify alternative hosts, such as fungi, were inconclusive, as fungal sequences were found inconsistently in the salmon tissue samples. The results of this study reinforce the need for further research to fully understand PMCV’s life cycle and potential alternative hosts and its whereabouts when it is not infecting the hearts of the Atlantic salmon. Full article

In the realm of plant biology, small RNAs (sRNAs) are imperative in the orchestration of gene expression, playing pivotal roles across a spectrum of developmental sequences and responses to environmental stressors. The biosynthetic cascade of sRNAs is characterized by an elaborate network of enzymatic pathways that meticulously process double-stranded RNA (dsRNA) precursors into sRNA molecules, typically 20 to 30 nucleotides in length. These sRNAs, chiefly microRNAs (miRNAs) and small interfering RNAs (siRNAs), are integral in guiding the RNA-induced silencing complex (RISC) to selectively target messenger RNAs (mRNAs) for post-transcriptional modulation. This regulation is achieved either through the targeted cleavage or the suppression of translational efficiency of the mRNAs. In plant development, sRNAs are integral to the modulation of key pathways that govern growth patterns, organ differentiation, and developmental timing. The biogenesis of sRNA itself is a fine-tuned process, beginning with transcription and proceeding through a series of processing steps involving Dicer-like enzymes and RNA-binding proteins. Recent advances in the field have illuminated the complex processes underlying the generation and function of small RNAs (sRNAs), including the identification of new sRNA categories and the clarification of their involvement in the intercommunication among diverse regulatory pathways. This review endeavors to evaluate the contemporary comprehension of sRNA biosynthesis and to underscore the pivotal role these molecules play in directing the intricate performance of plant developmental processes. Full article