Characterization of a novel gammapartitivirus infecting the phytopathogenic fungus Pyricularia oryzae.

In this study, we identified a novel double-strand RNA (dsRNA) mycovirus in Pyricularia oryzae, designated "Magnaporthe oryzae partitivirus 4" (MoPV4). The genome of MoPV4 consists of a dsRNA-1 segment encoding an RNA-dependent RNA polymerase (RdRP) and a dsRNA-2 segment encoding a capsid protein (CP). Phylogenetic analysis indicated that MoPV4 belongs to the genus Gammapartitivirus within family Partitiviridae. The particles of MoPV4 are isometric with a diameter of about 32.4 nm. Three-dimensional structure predictions indicated that the RdRP of MoPV4 forms a classical right-handed conformation, while the CP has a reclining-V shape.

CRISPR-Cas9 mediated dsRNase knockout improves RNAi efficiency in the fall armyworm.

Lepidopteran insects are refractory to RNA interference (RNAi) response, especially to orally delivered double-stranded RNA (dsRNA). High nuclease activity in the midgut lumen is proposed as one of the major reasons for RNAi insensitivity. We identified three dsRNase genes highly expressed in the midgut of fall armyworm (FAW), Spodoptera frugiperda. The genomic region harboring those three dsRNase genes was deleted using the CRISPR-Cas9-mediated genome editing method. A homozygous line with deletion of three dsRNase genes was produced. dsRNA degradation by midgut lumen contents of mutant larvae was lower than in wild-type larvae. Feeding dsRNA targeting the inhibitor of apoptosis (IAP) gene increased knockdown of the target gene and mortality in mutants compared to wild-type larvae. These results suggest that dsRNases in the midgut contribute to RNAi inefficiency in FAW. Formulations that protect dsRNA from dsRNase degradation may improve RNAi efficiency in FAW and other lepidopteran insects.

Shining light on DHX9: UV-induced stress granules illuminate protective mechanisms for daughter cell resilience.

In a recent article in Cell, Zhou et al. investigate the origins, composition, and biological consequences of UV-induced stress granules. They find that UV-induced stress granules are triggered by the formation of RNA-protein crosslinks, uniquely contain DHX9 as a marker, form during mitosis independently of translation repression, and are enriched in intron-containing RNAs and splicing factors. Moreover, UV-induced granules contain double-stranded RNA (dsRNA) and trigger a dsRNA response. This work identifies a mechanism for resolving UV-damaged RNA and broadens the types of cytosolic "stress granules" that form.

Complete genome sequence of a novel dsRNA virus from the phytopathogenic fungus Fusarium oxysporum.

Fusarium oxysporum is a widespread plant pathogen that causes fusarium wilt and fusarium root rot in many economically significant crops. Here, a novel dsRNA virus tentatively named "Fusarium oxysporum virus 1" (FoV1) was identified in F. oxysporum strain 3S-18. The genome of FoV1 is 2,944 nucleotides (nt) in length and contains two non-overlapping open reading frames (ORF1 and 2). The larger of these, ORF2, encodes an RNA-dependent RNA polymerase (RdRp) of 590 amino acids with a molecular mass of 67.52 kDa. ORF1 encodes a putative nucleocapsid protein consisting of 134 amino acids with a molecular mass of 34.25 kDa. The RdRp domain of FoV1 shares 60.00% to 84.24% sequence identity with non-segmented dsRNA viruses. Phylogenetic analysis further suggested that FoV1 is a new member of the proposed genus "Unirnavirus" accommodating unclassified monopartite dsRNA viruses.

Complete genome sequence of a novel alternavirus isolated from the phytopathogenic fungus Colletotrichum fioriniae.

A novel double-strand RNA (dsRNA) mycovirus, named "Colletotrichum fioriniae alternavirus1" (CfAV1), was isolated from the strain CX7 of Colletotrichum fioriniae, the causal agent of walnut anthracnose. The complete genome of CfAV1 is composed of three dsRNA segments: dsRNA1 (3528 bp), dsRNA2 (2485 bp), and dsRNA3 (2481 bp). The RNA-dependent RNA polymerase (RdRp) is encoded by dsRNA1, while both dsRNA2 and dsRNA3 encode hypothetical proteins. Based on multiple sequence alignments and phylogenetic analysis, CfAV1 is identified as a new member of the family Alternaviridae. This is the first report of an alternavirus that infects the phytopathogenic fungus C. fioriniae.

First detection of mycoviruses in Gnomoniopsis castaneae suggests a putative horizontal gene transfer event between negative-sense and double-strand RNA viruses.

Gnomoniopsis castaneae is an ascomycetous fungus mainly known as a major pathogen of chestnut causing nut rots, although it is often found as an endophyte in chestnut tissues. To date, no virus has been reported as associated with to this fungus. Here, a collection of G. castaneae isolates from several European countries was screened to detect mycoviruses infecting the fungus: for the first time we report the identification and prevalence of mitovirus Gnomoniopsis castaneae mitovirus 1 (GcMV1) and the chrysovirus Gnomoniopsis castaneae chrysovirus 1 (GcCV1). Interestingly, we provide evidence supporting a putative horizontal gene transfer between members of the phyla Negarnaviricota and Duplornaviricota: a small putative protein of unknown function encoded on the RNA3 of GcCV1 (Chrysoviridae) has homologs in the genome of viruses of the family Mymonaviridae.

Characterization of the RNA Mycovirome Associated with Grapevine Fungal Pathogens: Analysis of Mycovirus Distribution and Their Genetic Variability within a Collection of Botryosphaeriaceae Isolates.

Botryosphaeriaceae are fungi involved in the decay of various woody species, including the grapevine, leading to significant production losses. This fungal family is largely ubiquitous, and seven species of Botryosphaeriaceae have been identified in French vineyards, with variable levels of aggressiveness, both in vitro and in planta. Mycoviruses can impact the life traits of their fungal hosts, including aggressiveness, and are one of the factors influencing fungal pathogenicity. In this study, the RNA mycovirome of fifteen Botryosphaeriaceae isolates was characterized through the high-throughput sequencing of double-stranded RNA preparations from the respective samples. Eight mycoviruses were detected, including three potential novel species in the Narnaviridae family, as well as in the proposed Mycobunyaviridae and Fusagraviridae families. A large collection of Botryosphaeriaceae isolates was screened using RT-PCR assays specific for 20 Botryosphaeriaceae-infecting mycoviruses. Among the mycoviruses detected, some appeared to be specialists within a single host species, while others infected isolates belonging to multiple Botryosphaeriaceae species. This screening allowed us to conclude that one-third of the Botryosphaeriaceae isolates were infected by at least one mycovirus, and a significant proportion of isolates (43.5%) were found to be coinfected by several viruses, with very complex RNA mycoviromes for some N. parvum isolates.

Exogenous Application of dsRNA for Protection against Tomato Leaf Curl New Delhi Virus.

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed.

Multifaceted roles of RNA editing enzyme ADAR1 in innate immunity.

Innate immunity must be tightly regulated to enable sensitive pathogen detection while averting autoimmunity triggered by pathogen-like host molecules. A hallmark of viral infection, double-stranded RNAs (dsRNAs) are also abundantly encoded in mammalian genomes, necessitating surveillance mechanisms to distinguish "self" from "nonself." ADAR1, an RNA editing enzyme, has emerged as an essential safeguard against dsRNA-induced autoimmunity. By converting adenosines to inosines (A-to-I) in long dsRNAs, ADAR1 covalently marks endogenous dsRNAs, thereby blocking the activation of the cytoplasmic dsRNA sensor MDA5. Moreover, beyond its editing function, ADAR1 binding to dsRNA impedes the activation of innate immune sensors PKR and ZBP1. Recent landmark studies underscore the utility of silencing ADAR1 for cancer immunotherapy, by exploiting the ADAR1-dependence developed by certain tumors to unleash an antitumor immune response. In this perspective, we summarize the genetic and mechanistic evidence for ADAR1's multipronged role in suppressing dsRNA-mediated autoimmunity and explore the evolving roles of ADAR1 as an immuno-oncology target.

Limited dsRNA editing impedes leukemia stem cells.

Understanding the mechanisms underlying the generation and maintenance of leukemia stem cells (LSCs) is crucial for the development of effective therapies against T cell acute lymphoblastic leukemia (T-ALL). In a recent study, Rivera et al. discovered that elevated adenosine deaminase acting on RNA (ADAR)-1-mediated RNA editing is a distinguishing feature of T-ALL relapse, and that ADAR1 suppresses apoptosis triggered by the double-stranded (ds)RNA-sensing pathway.

Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells.

Detection of viral double-stranded RNA (dsRNA) is an important component of innate immunity. However, many endogenous RNAs containing double-stranded regions can be misrecognized and activate innate immunity. The IFN-inducible ADAR1-p150 suppresses dsRNA sensing, an essential function for adenosine deaminase acting on RNA 1 (ADAR1) in many cancers, including breast. Although ADAR1-p150 has been well established in this role, the functions of the constitutively expressed ADAR1-p110 isoform are less understood. We used proximity labeling to identify putative ADAR1-p110-interacting proteins in breast cancer cell lines. Of the proteins identified, the RNA helicase DHX9 was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of the dsRNA sensor PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Together, these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways. SIGNIFICANCE: These findings implicate DHX9 as a suppressor of dsRNA sensing. In some cell lines, loss of DHX9 alone is sufficient to cause activation of dsRNA sensing pathways, while in other cell lines DHX9 functions redundantly with ADAR1 to suppress pathway activation.

RNA is a pro-apoptotic target of cisplatin in cancer cell lines and C. elegans.

Cisplatin not only targets DNA but also RNA. However, it is largely unknown whether platinated RNA (Pt-RNA) causes apoptosis and thus contributes to the cytotoxic effects of cisplatin. Consequently, cellular RNA was isolated from HepG2 and LS180 cells, exposed to cisplatin, and the resulting Pt-RNA (20 ng Pt/µg RNA) was transfected into these cancer cell lines or used to treat an apoptosis reporter Caenorhabditis elegans (C. elegans) strain (MD701, expressing CED-1::GFP). Cellular and molecular effects of Pt-RNA were evaluated by luminogenic caspase 3/7 assays, PCR array analysis, and fluorescence microscopy-based quantification of apoptosis in C. elegans gonads. Assuming RNA cross-linking (pseudo double-stranded RNA), the contribution of the Toll-like receptor 3 (TLR3, a sensor of double-stranded RNA) to apoptosis induction in cancer cell lines was investigated by pharmacological TLR3 inhibition and overexpression. In contrast to controls, Pt-RNA significantly enhanced apoptosis in C. elegans (2-fold) and in the cancer cell lines (2-fold to 4-fold). TLR3 overexpression significantly enhanced the pro-apoptotic effects of Pt-RNA in HepG2 cells. TLR3 inhibition reduced the pro-apoptotic effects of Pt-RNA and cisplatin, but not of paclitaxel (off-target control). Gene expression analysis showed that Pt-RNA (but not RNA) significantly enhanced the mRNA levels of nuclear factor kappa B subunit 2 and interleukin-8 in HepG2 cells, suggesting that Pt-RNA is a damage-associated molecular pattern that additionally causes pro-inflammatory responses. Together, this data suggests that not only DNA but also cellular RNA is a functionally relevant target of cisplatin, leading to pro-apoptotic and immunogenic effects.

DsRNA-based pesticides: Considerations for efficiency and risk assessment.

In view of the ongoing climate change and the ever-growing world population, novel agricultural solutions are required to ensure sustainable food supply. Microbials, natural substances, semiochemicals and double stranded RNAs (dsRNAs) are all considered potential low risk pesticides. DsRNAs function at the molecular level, targeting specific regions of specific genes of specific organisms, provided that they share a minimal sequence complementarity of approximately 20 nucleotides. Thus, dsRNAs may offer a great alternative to conventional chemicals in environmentally friendly pest control strategies. Any low-risk pesticide needs to be efficient and exhibit low toxicological potential and low environmental persistence. Having said that, in the current review, the mode of dsRNA action is explored and the parameters that need to be taken into consideration for the development of efficient dsRNA-based pesticides are highlighted. Moreover, since dsRNAs mode of action differs from those of synthetic pesticides, custom-made risk assessment schemes may be required and thus, critical issues related to the risk assessment of dsRNA pesticides are discussed here.

The complete genome sequences of a negative single-stranded RNA virus and a double-stranded RNA virus coinfecting the entomopathogenic fungus Beauveria bassiana Vuillemin.

Beauveria bassiana Vuillemin is an entomopathogenic fungus that has been developed as a biological insecticide. B. bassiana can be infected by single or multiple mycoviruses, most of which are double-stranded RNA (dsRNA) viruses, while infections with single-stranded RNA (ssRNA) viruses, especially negative single-stranded RNA (-ssRNA) viruses, have been observed less frequently. In the present study, we sequenced and analyzed the complete genomes of two new different mycoviruses coinfecting a single B. bassiana strain: a -ssRNA virus which we have named "Beauveria bassiana negative-strand RNA virus 1" (BbNSRV1), and a dsRNA virus, which we have named "Beauveria bassiana orthocurvulavirus 1" (BbOCuV1). The genome of BbNSRV1 consists of a single segment of negative-sense, single-stranded RNA with a length of 6169 nt, containing a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) with 1949 aa (220.1 kDa). BLASTx analysis showed that the RdRp had the highest sequence similarity (59.79%) to that of Plasmopara viticola lesion associated mononegaambi virus 2, a member of the family Mymonaviridae. This is the first report of a -ssRNA mycovirus infecting B. bassiana. The genome of BbOCuV1 consists of two dsRNA segments, 2164 bp and 1765 bp in length, respectively, with dsRNA1 encoding a protein with conserved RdRp motifs and 70.75% sequence identity to the putative RdRp of the taxonomically unassigned mycovirus Fusarium graminearum virus 5 (FgV5), and the dsRNA2 encoding a putative coat protein with sequence identity 64.26% to the corresponding protein of the FgV5. Phylogenetic analysis indicated that BbOCuV1 belongs to a taxonomically unassigned group of dsRNA mycoviruses related to members of the families Curvulaviridae and Partitiviridae. Hence, it might be the member of a new family that remains to be named and formally recognized.

Complete genome sequence of a novel gammapartitivirus from Penicillium oxalicum RCEF7482.

Penicillium oxalicum, an important biocontrol fungus in China, has been a subject of extensive study due to its role in combating various pathogenic fungi. Despite the prevalence of mycoviruses with double-stranded (ds) RNA genomes in filamentous fungi, there has been no screening of mycoviruses in P. oxalicum. In this report, we describe the identification and characterization of a novel dsRNA virus isolated from P. oxalicum, designated as "Penicillium oxalicum partitivirus 1" (PoPV1). The genome of PoPV1 consists of two dsRNA segments, dsRNA1 (1,770 bp) and dsRNA2 (1,584 bp), each containing a single open reading frame (ORF): ORF1 and ORF2. Comparative analysis revealed that the RdRp and CP amino acid sequences of PoPV1 share the highest identity (89.18% and 73.97%, respectively) with those of Penicillium aurantiogriseum partitivirus 1 (PaPV1). Motif analysis based on RdRp amino acid sequences places PoPV1 in the genus Gammapartitivirus within the family Partitiviridae, with a distinctive motif VI (R/KV/ILGDD). Phylogenetic analysis further established a close relationship of PoPV1 to PaPV1, forming a unique clade among the gammapartitiviruses. Consequently, we propose that Penicillium oxalicum partitivirus 1 represents a new species in the genus Gammapartitivirus. This is the first report of a dsRNA virus in P. oxalicum.

A novel alternavirus with three dsRNA segments from Fusarium pseudograminearum, the pathogen of Fusarium crown rot in wheat.

Three dsRNA segments were detected in Fusarium pseudograminearum strain CF14029, a pathogen causing Fusarium crown rot in China. Characterization and sequence analysis confirmed that these dsRNA sequences originated from the same virus. The viral genome consists of three dsRNA segments: dsRNA1 (3,560 nt in length), encoding an RNA-dependent RNA polymerase (RdRp), dsRNA2 (2,544 nt in length), encoding a hypothetical protein, and dsRNA3 (2,478 nt in length), encoding a putative coat protein (CP). Phylogenetic analysis based on the RdRp and CP amino acid sequences revealed a high degree of similarity of this virus to members of the genus Alternavirus, family Alternaviridae, isolated from other Fusarium fungi. As a novel member of the genus Alternavirus, this virus was provisionally named "Fusarium pseudograminearum alternavirus 1" (FpgAV1). Like other alternaviruses found in Fusarium species, the positive-sense strand of each genomic dsRNA of FpgAV1 possesses a poly(A) tail and a distinctive 5'-terminal octamer sequence (5'-GCT GTG TG-3'). This is the first report of the genomic sequence of an alternavirus identified in F. pseudograminearum.

Oral delivery of bacteria expressing wsv108 gene-specific dsRNA protects shrimp from white spot syndrome virus (WSSV) infection.

Double-stranded RNA (dsRNA) can specifically inhibit gene expression by RNA interference and has important application potential in animal disease control. White spot syndrome virus (WSSV) is one of the most harmful pathogens in shrimp aquaculture, causing huge economic losses every year. In this study, we investigated the function of the WSSV-encoded wsv108 protein. We demonstrated that wsv108 could promote apoptosis by interacting with heat shock protein 70 (HSP70) and enhancing the expression of multiple apoptosis-related genes. Silencing of wsv108 gene by injection with specific dsRNA prepared by in vitro transcription significantly increased the survival rate of WSSV-infected shrimp and reduced the viral load in tissues, suggesting that wsv108 is important for WSSV pathogenicity. Based on this, we expressed the wsv108 specific dsRNA in engineered Escherichia coli. Oral feeding of this bacterium could inhibit the expression of wsv108, increase the survival rate of WSSV-infected shrimp, and decrease the viral load of WSSV in tissues. Therefore, this study developed a new method for treatment of WSSV disease by oral administration of bacterially expressed dsRNA against a novel therapeutic target molecule, which could be a potential candidate strategy for WSSV control in aquaculture.

Mechanistic exploration of autophagy and aging by RNA interference.

Cellular senescence is a cellular process with organismal impact that is mechanistically counterbalanced to a certain extent by frequent episodes of autophagy. Here we describe a detailed, automation-compatible method for the use of RNA-interference (RNAi; also called post-transcriptional gene silencing (PTGS))-mediated silencing of autophagy related protein-coding gene expression. RNAi is a conserved biological response to double-stranded RNA that mediates resistance to endogenous parasites and exogenous pathogenic nucleic acids. RNAi mediated by short interfering RNA (siRNA) is widely used for gene function analysis. The accurate use of RNAi for the inference of gene function necessitates that both specificity and efficacy of the siRNA-mediated knockdown are monitored. In this manuscript, we exemplify these crucial steps employing siRNAs targeting the autophagy and lysosomal biogenesis associated transcription factor TFE3 and validate their specificity on protein and mRNA level.

Graphene Quantum Dots (GQD)-Mediated dsRNA Delivery for the Control of Fusarium Head Blight Disease in Wheat.

Graphene quantum dots (GQDs), a class of fluorescent carbon materials, have displayed significant potential in various fields such as energy devices, catalysis, sensing, bioimaging, and drug delivery. Because of their extremely small size, generally less than 100 nm, they also have tremendous potential in plant science research, especially for the delivery of nucleic acids, breaking the barrier of cell walls. In this study, we synthesized GQDs with a size range of 2-5 nm, characterized them, and surface-functionalized them with branched polyethylenimine (bPEI). We then used the surface-functionalized GQDs as carriers to deliver double-stranded RNA (dsRNA) that target two growth-and-development-related genes in Fusarium graminearum─the causative organism of the Fusarium head blight disease of wheat. The successful binding of dsRNA to GQDs-bPEIs was demonstrated through gel-shifting assays, showcasing the potential for efficient dsRNA delivery. We designed dsRNAs targeting the MGV1 and RAS1 genes of F. graminearum by using the pssRNAit pipeline, ensuring high specificity and no off-target effects. The coding sequences of the designed dsRAS1 and dsMGV1 were cloned into the L4440 vector and transformed into the Escherichia coli HT115 strain for dsRNA production. Fungal culture analysis revealed that the inclusion of dsRNAs in potato dextrose agar (PDA) media effectively slowed down the growth. Exogenous spraying experiments both in plate cultures and in intact wheat spikes demonstrated that the dsRNA:GQDs-bPEIs treatment was more effective in restricting fungal mycelium growth or the number of infected spikelets compared to naked dsRNA treatment. Our study demonstrates the promising potential of graphene quantum dots as carriers for dsRNA-based fungal disease management in wheat and other crops.

Satellite double-stranded RNA induces mesenchymal transition in pancreatic cancer by regulating alternative splicing.

Human satellite II (HSATII), composed of tandem repeats in pericentromeric regions, is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of repetitive elements in cancer tissues can facilitate incidental dsRNA formation; however, it remains controversial whether dsRNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the double-stranded formation of HSATII RNA and explored its molecular function. The overexpression of double-stranded HSATII (dsHSATII) RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, spermatid perinuclear RNA-binding protein (STRBP), which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. The mesenchymal transition of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with epithelial-mesenchymal transition (EMT). We also confirmed that isoform switching of CLSTN1, driven by dsHSATII overexpression or STRBP depletion, induced EMT-like morphological changes. These findings reveal a novel tumor-promoting function of dsHSATII RNA, inducing EMT-like changes and cell invasiveness, thus enhancing our understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors.