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PubMed2026

A small viral protein suppresses immune amplification by two distinct mechanisms.

Diverse viral suppressors of RNA interference (RNAi) and RNA silencing (VSRs) interact directly with core protein and/or RNA components of the host RNAi pathway. However, the specific counter-defense function of any VSR biochemical activity is fully validated only when it is shown as essential for viral infection in the wild-type but not mutant hosts defective in antiviral RNAi. Here, we investigated the role of VSR activities for direct binding to small-interfering RNA duplexes (siRNA), long double-stranded RNA (dsRNA), or RNA-dependent RNA polymerase 1 (RDR1) during plant infection by wild-type and mutant cucumber mosaic virus (CMV), a positive-strand RNA virus expressing the 110-residue 2b protein as its VSR. We demonstrate that a C-terminally truncated 2b mutant (2b1-93) active in direct binding to siRNA and dsRNA, but not RDR1, was able to suppress the amplification of virus-derived siRNAs (vsiRNA) and antiviral RNAi mediated by RDR6, but not RDR1. By contrast, an N-terminally truncated 2b mutant (2b18-110) inactive in direct binding to siRNA or dsRNA was able to suppress vsiRNA amplification and antiviral RNAi mediated by RDR1, but not RDR6, and was less effective to promote systemic CMV infection and disease development than 2b1-93. Together, our results show that whereas RDR1 suppression requires direct binding of VSR-2b to RDR1, but not siRNA or dsRNA, RDR6 suppression depends on direct binding to siRNA and dsRNA, but not RDR1. Therefore, CMV, through its VSR-2b, suppresses two parallel vsiRNA amplification pathways by distinct molecular mechanisms, and this unique property may account for the unusually wide host range of CMV.IMPORTANCEHost amplification of antiviral immunity is essential for robust control of viral infections. However, little is known about the mechanisms that viruses have evolved to suppress immune amplification in plants. Here, we characterized whole plant infection by cucumber mosaic virus (CMV) with its viral suppressor of RNA interference (RNAi) mutated to become inactive in direct binding to small-interfering RNA duplexes (siRNA), long double-stranded RNA (dsRNA), or RNA-dependent RNA polymerase 1 (RDR1). We demonstrate maximal suppression of both RDR1- and RDR6-mediated antiviral RNAi amplification by the CMV 2b protein, a viral suppressor of RNAi (VSR). Notably, whereas RDR1 suppression requires direct binding of 2b to RDR1 but not siRNA or dsRNA, RDR6 suppression depends on direct binding to siRNA and dsRNA, but not RDR1. Our findings reveal a novel counter-defense strategy evolved by a wide host range positive-strand RNA virus to suppress two pathways of immune amplification by distinct mechanisms.

Cucumovirus↗
PubMed1995

Use of a polyethylene glycol-peptide conjugate in a competition gel shift assay for screening potential antagonists of HIV-1 Tat protein binding to TAR RNA.

Interference of binding of Tat protein to TAR RNA in HIV-1-infected cells may be a useful therapeutic strategy for AIDS. An electrophoretic assay to screen potential low-molecular-weight (< 2 kDa) Tat antagonists has been established. A radiolabeled TAR RNA fragment (delta TAR) is retarded in mobility when bound by a Tat peptide-polyethylene glycol conjugate (Tat-PEG), which is used in place of the Tat protein. The assay determines the ability of a potential antagonist to compete with Tat-PEG for binding to delta TAR, as measured by interference with the gel shift of delta TAR. To discriminate between specific and nonspecific interactions, the assay is done in the absence or the presence of a 250-fold molar excess of tRNA.

Amino Acid Sequence↗
PubMed2000

Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana.

We investigated the potential of double-stranded RNA interference (RNAi) with gene activity in Arabidopsis thaliana. To construct transformation vectors that produce RNAs capable of duplex formation, gene-specific sequences in the sense and antisense orientations were linked and placed under the control of a strong viral promoter. When introduced into the genome of A. thaliana by Agrobacterium-mediated transformation, double-stranded RNA-expressing constructs corresponding to four genes, AGAMOUS (AG), CLAVATA3, APETALA1, and PERIANTHIA, caused specific and heritable genetic interference. The severity of phenotypes varied between transgenic lines. In situ hybridization revealed a correlation between a declining AG mRNA accumulation and increasingly severe phenotypes in AG (RNAi) mutants, suggesting that endogenous mRNA is the target of double-stranded RNA-mediated genetic interference. The ability to generate stably heritable RNAi and the resultant specific phenotypes allows us to selectively reduce gene function in A. thaliana.

AGAMOUS Protein, Arabidopsis↗
PubMed1982

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. II. Evidence that large N-terminal amber fragments of the beta subunit interfere with RNA polymerase function.

A collection of 95 independent, spontaneously-occurring mutants carrying amber lesions that affect expression of the beta gene, rpoB, has been isolated (see accompanying paper (Nene and Glass 1982)). Certain rpoB amber mutations act in trans, preventing a functional allele present on an F' plasmid from acting at high temperature. Two such temperature-sensitive rpoB(Am) strains are shown to produce large, N-terminal amber fragments. The possibility that these truncated polypeptides are the cause of this transdominant conditional-lethal phenotype is supported by analysis of fragment levels in thermoresistant survivors: the nonsense fragments are degraded at a significantly faster rate (half-lives 1.4- to 2.6-fold reduced) in Ts+ derivatives likely to carry second-site mutations within rpoB. We suggest that the beta fragments interfere with RNA polymerase function by interacting with one or more of the polymerase subunits.

DNA-Directed RNA Polymerases↗
PubMed2000

Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes.

Double-stranded RNA interference (RNAi) is an effective method for disrupting expression of specific genes in Caenorhabditis elegans and other organisms. Applications of this reverse-genetics tool, however, are somewhat restricted in nematodes because introduced dsRNA is not stably inherited. Another difficulty is that RNAi disruption of late-acting genes has been generally less consistent than that of embryonically expressed genes, perhaps because the concentration of dsRNA becomes lower as cellular division proceeds or as developmental time advances. In particular, some neuronally expressed genes appear refractory to dsRNA-mediated interference. We sought to extend the applicability of RNAi by in vivo expression of heritable inverted-repeat (IR) genes. We assayed the efficacy of in vivo-driven RNAi in three situations for which heritable, inducible RNAi would be advantageous: (i) production of large numbers of animals deficient for gene activities required for viability or reproduction; (ii) generation of large populations of phenocopy mutants for biochemical analysis; and (iii) effective gene inactivation in the nervous system. We report that heritable IR genes confer potent and specific gene inactivation for each of these applications. We suggest that a similar strategy might be used to test for dsRNA interference effects in higher organisms in which it is feasible to construct transgenic animals, but impossible to directly or transiently introduce high concentrations of dsRNA.

Animals↗
PubMed2025

Microbe-induced gene silencing of fungal gene confers efficient resistance against Fusarium graminearum in maize.

UNLABELLED: Small RNAs (sRNAs), the main effectors of RNA interference (or RNA silencing, RNAi), mediate cell-autonomous and non-cell-autonomous gene silencing. The discoveries of trans-kingdom RNAi and interspecies RNAi have accelerated the development of RNAi-based crop protection technologies. Recently, based on interspecies RNAi, a practical technology termed microbe-induced gene silencing (MIGS) without the need of host genetic modification is developed for crop protection against Verticillium dahliae and Fusarium oxysporum in cotton and rice plants. In this study, we utilized MIGS technology to protect maize against Fusarium graminearum, which is responsible for maize stalk rot. An RNAi-engineered Trichoderma harzianum strain, Th-FgPmt2i, was exploited to generate double-stranded RNAs (dsRNAs) to trigger the silencing of the FgPTM2 gene. Our data verify that sRNAs generated from Th-FgPmt2i can silence the FgPMT2 gene via translational inhibition in F. graminearum. We further demonstrated that Th-FgPmt2i has a stronger capacity than does the T. harzianum chassis for protection of maize against F. graminearum. Coupled with our studies on crop protection against V. dahliae and F. oxysporum, our findings reveal that MIGS can be exploited to protect various crops against distinct fungal pathogens and has extensive applicability. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-025-00212-9.

Fusarium graminearum↗
PubMed2001

Genetic interference in protozoa.

RNA interference first described in Caenorhabditis elegans and transgene-induced post-transcriptional gene silencing first described in plants and fungi now appear as different means of activating a conserved and ancient mechanism that can protect genomes against viruses and transposons and perhaps also control expression of endogenous genes. We present here similar genetic interference phenomena in highly divergent protozoa, Trypanosoma and Paramecium, and look ahead to what contribution these microorganisms could bring to this fast-moving area.

Animals↗
PubMed1993

Poly(A) signals and transcriptional pause sites combine to prevent interference between RNA polymerase II promoters.

Transcriptional termination by RNA polymerase II at the 3' end of genes encoding poly(A)+ mRNAs is thought to require two distinct cis-active elements: a functional poly(A) signal and a downstream transcriptional pause site. An important requirement for efficient termination is to prevent transcriptional interference of downstream-located promoters. We have therefore investigated whether these two elements, individually or in combination, can prevent transcriptional interference of RNA polymerase II-activated promoters. For this purpose, we constructed an expression plasmid containing two tandem retroviral long terminal repeats (LTRs) derived from HIV-1. When transfected into HeLa cells, this construct resulted in transcriptional interference of the LTR promoters. Using this assay, we were able to show that a single poly(A) signal was able to protect an otherwise occluded promoter. This effect depended on the RNA-processing strength of the poly(A) signal. Furthermore, transcriptional pause sites provided adequate protection against promoter occlusion even when tested alone. Finally, a combined element consisting of a poly(A) signal followed by a pause site was more efficient in promoter protection than either element on its own. These results indicate that an interference-blocking element can take various forms: a poly(A) signal, a transcriptional pause site or a combination of both.

HIV Long Terminal Repeat↗
PubMed1999

RNA-Mediated interference of a cdc25 homolog in Caenorhabditis elegans results in defects in the embryonic cortical membrane, meiosis, and mitosis.

The CDC25 dual-specificity phosphatase family has been shown to play a key role in cell cycle regulation. The phosphatase activity of CDC25 drives the cell cycle by removing inhibitory phosphates from cyclin-dependent kinase/cyclin complexes. Although the regulation of CDC25 phosphatase activity has been elucidated both biochemically and genetically in other systems, the role of this enzyme during development is not well understood. To examine the expression pattern and function of CDC25 in Caenorhabditis elegans, we characterized a cdc25 homolog, cdc-25.1, during early embryonic development. The CDC-25.1 protein localizes to oocytes, embryonic nuclei, and embryonic cortical membranes. When the expression of CDC-25.1 was disrupted by RNA-mediated interference, the anterior cortical membrane of fertilized eggs became very fluid during meiosis and subsequent mitotic cell cycles. Mispositioning of the meiotic spindle, defects in polar body extrusion and chromosome segregation, and abnormal cleavage furrows were also observed. We conclude that CDC-25.1 is required for a very early developmental process-the proper completion of meiosis prior to embryogenesis.

Animals↗
PubMedDate not supplied

Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects.

Gene product distribution is often used to infer developmental similarities and differences in animals with evolutionarily diverse body plans. However, to address commonalties of developmental mechanisms, what is really needed is a method to assess and compare gene function in divergent organisms. This requires mutations eliminating gene function. Such mutations are often difficult to obtain, even in organisms amenable to genetic analysis. To address this issue we have investigated the use of double-stranded RNA interference to phenocopy null mutations. We show that RNA interference can be used to phenocopy mutations of the Deformed orthologues in Drosophila and Tribolium. We discuss the possible use of this technique for comparisons of developmental mechanisms in organisms with differing ontogenies.

Animals↗
PubMed1999

The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila.

Wingless (Wg) is a member of the Wnt family of growth factors, secreted proteins that control proliferation and differentiation during development. Studies in Drosophila have shown that responses to Wg require cell-surface heparan sulphate, a glycosaminoglycan component of proteoglycans. These findings suggest that a cell-surface proteoglycan is a component of a Wg/Wnt receptor complex. We demonstrate here that the protein encoded by the division abnormally delayed (dally) gene is a cell-surface, heparan-sulphate-modified proteoglycan. dally partial loss-of-function mutations compromise Wg-directed events, and disruption of dally function with RNA interference produces phenotypes comparable to those found with RNA interference of wg or frizzled (fz)/Dfz2. Ectopic expression of Dally potentiates Wg signalling without altering levels of Wg and can rescue a wg partial loss-of-function mutant. We also show that dally, a regulator of Decapentaplegic (Dpp) signalling during post-embryonic development, has tissue-specific effects on Wg and Dpp signalling. Dally can therefore differentially influence signalling mediated by two growth factors, and may form a regulatory component of both Wg and Dpp receptor complexes.

Animals↗
PubMed1991

Non-replicating deletion mutants of brome mosaic virus RNA-2 interfere with viral replication.

Naturally occurring defective interfering RNAs (DI-RNAs) and satellite RNAs greatly reduce the accumulation of their helper virus in vivo, but often modulate symptom expression in an unpredictable manner. Deletion mutants Nc/S, Na/M and Sa/Nc + M/S, derived from brome mosaic virus (BMV) RNA-2, failed to replicate when co-inoculated with BMV RNAs-1 and -2 to barley protoplasts. However, the inoculum RNA corresponding to these deletion mutants was extremely stable and could have been mistaken for plus-strand progeny had minus-strand progeny analysis been omitted. These results accentuate the need for such tests in evaluating the ability of mutant viral sequences to replicate. One of the mutants, Nc/S, effectively interfered with the accumulation of BMV RNAs-1 and -2 in barley protoplasts. This non-replicating interfering RNA was termed NRI RNA-2 Nc/S. When present with RNAs-1 and -2 at low inoculum amounts (1 microgram), NRI RNA-2 Nc/S reduced replication of RNA-2, the parental RNA, by 63% and preferentially interfered with minus-strand RNA accumulation. At higher levels (4 micrograms), it completely displaced replication of both RNAs-1 and -2. Mutations eliminating translation of a truncated p2a protein from NRI RNA-2 Nc/S did not alleviate the interference effect, demonstrating that a defective replicase protein was not responsible for the decreased accumulation of genomic RNA. At an NRI RNA: genomic RNA inoculum molar ratio of 1:1, NRI RNA-2 Nc/S reduced the accumulation of all helper virus RNAs by 55%. Since this reduction was seen for both wild-type RNA-3 and delta SGP RNA-3, a deletion mutant of RNA-3 that lacks the subgenomic promoter necessary for coat protein expression, it was evident that the effective interference mediated by NRI RNA-2 Nc/S was not mitigated by encapsidation. The ability of the NRI RNAs to mimic satellite DI RNAs in depressing helper virus replication suggests that their expression in transgenic plants may provide a new and widely applicable approach for inducing resistance to viral infection.

Blotting, Northern↗
PubMed1995

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.

Base Sequence↗
PubMed2021

Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake.

Recent discoveries show that fungi can take up environmental RNA, which can then silence fungal genes through environmental RNA interference. This discovery prompted the development of Spray-Induced Gene Silencing (SIGS) for plant disease management. In this study, we aimed to determine the efficacy of SIGS across a variety of eukaryotic microbes. We first examined the efficiency of RNA uptake in multiple pathogenic and non-pathogenic fungi, and an oomycete pathogen. We observed efficient double-stranded RNA (dsRNA) uptake in the fungal plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, Aspergillus niger and Verticillium dahliae, but no uptake in Colletotrichum gloeosporioides, and weak uptake in a beneficial fungus, Trichoderma virens. For the oomycete plant pathogen, Phytophthora infestans, RNA uptake was limited and varied across different cell types and developmental stages. Topical application of dsRNA targeting virulence-related genes in pathogens with high RNA uptake efficiency significantly inhibited plant disease symptoms, whereas the application of dsRNA in pathogens with low RNA uptake efficiency did not suppress infection. Our results have revealed that dsRNA uptake efficiencies vary across eukaryotic microbe species and cell types. The success of SIGS for plant disease management can largely be determined by the pathogen's RNA uptake efficiency.

Ascomycota↗
PubMed2001

Comparative insect developmental genetics: phenotypes without mutants.

The last decade has seen a dramatic increase in interest in the extent to which morphological evolution depends on changes in regulatory pathways. Insects provide a fertile ground for study because of their diversity and our high level of understanding of the genetic regulation of development in Drosophila melanogaster. However, comparable genetic approaches are presently possible in only a small number of non-Drosophilid insects. In a recent paper, Hughes and Kaufman have used a new methodology, RNA interference, in the milkweed bug, Oncopeltus fasciatus, to phenocopy the effects of mutations in Hox genes. RNA interference involves the injection of double-stranded RNA of the same sequence as the relevant mRNA resulting in a depletion of that transcript. Hughes and Kaufman focused on the gnathal segments, which elaborate specialized appendages important to feeding. Their results indicate that gnathal adaptations in this bug are correlated with changes in Hox gene functions and interactions.

Animals↗
PubMed2000

Requirement of V-ATPase for ovulation and embryogenesis in Caenorhabditis elegans.

Immunofluorescence analysis indicated that VHA-11, the C subunit of Caenorhabditis elegans V-ATPase, was localized in dot-like structures around the nuclei of early embryonic cells and was also detected in embryonic intestinal cells after comma stage. Vital staining with acridine orange showed that the intestinal cells had acidic compartments generated by V-ATPase, consistent with the intracellular localization of VHA-11. RNA interference could efficiently silence vha-11 gene expression: introduction of vha-11 double strand RNA led to embryonic lethality. Worms injected with the vha-11 double strand RNA produced embryos that became lethal. The development of embryos was arrested at various stages. However, their numbers gradually decreased, and the worms eventually became sterile due to the failure of ovulation. Similar results were obtained for RNA interference of the V-ATPase proteolipid genes. These results suggest that V-ATPases, and thus inside-acidic organelles, are required for ovulation and embryogenesis.

Animals↗
PubMed2000

The conserved nuclear receptor Ftz-F1 is required for embryogenesis, moulting and reproduction in Caenorhabditis elegans.

BACKGROUND: Nuclear receptors are essential players in the development of all metazoans. The nematode Caenorhabditis elegans possesses more than 200 putative nuclear receptor genes, several times more than the number known in any other organism. Very few of these transcription factors are conserved with components of the steroid response pathways in vertebrates and arthropods. Ftz-F1, one of the evolutionarily oldest nuclear receptor types, is required for steroidogenesis and sexual differentiation in mice and for segmentation and metamorphosis in Drosophila. RESULTS: We employed two complementary approaches, direct mutagenesis and RNA interference, to explore the role of nhr-25, a C. elegans ortholog of Ftz-F1. Deletion mutants show that nhr-25 is essential for embryogenesis. RNA interference reveals additional requirements throughout the postembryonic life, namely in moulting and differentiation of the gonad and vulva. All these defects are consistent with the nhr-25 expression pattern, determined by in situ hybridization and GFP reporter activity. CONCLUSIONS: Our data link the C. elegans Ftz-F1 ortholog with a number of developmental processes. Significantly, its role in the periodical replacement of cuticle (moulting) appears to be evolutionarily shared with insects and thus supports the monophyletic origin of moulting.

Animals↗