Molecular characterization of two endogenous double-stranded RNAs in rice and their inheritance by interspecific hybrids

We completely sequenced 13,936 nucleotides (nt) of a double-stranded RNA (dsRNA) of wild rice (W-dsRNA). A single long open reading frame (13,719 nt) containing the conserved motifs of RNA-dependent RNA polymerase and RNA helicase was located in the coding strand. The identity between entire nucleotide sequence of W-dsRNA and that of the dsRNA of temperate japonica rice (J-dsRNA, 13,952 nt) was 75.5%. A site-specific discontinuity (nick) was identified at nt 1,197 from the 5' end of the coding strand of W-dsRNA. This nick is also located at nt 1,211 from the 5' end in the coding strand of J-dsRNA. The dsRNA copy number was increased more than 10-fold in pollen grains of both rice plants. This remarkable increase may be responsible for the highly efficient transmission of J-dsRNA via pollen that we already reported. J-dsRNA and W-dsRNA were also efficiently transmitted to interspecific F1 hybrids. Seed-mediated dsRNA transmission to F2 plants was also highly efficient when the maternal parent was wild rice. The efficiency of dsRNA transmission to F2 plants was reduced when the maternal parent was temperate japonica rice; however, the reduced rates in F2 plants were returned to high levels in F3 plants.     

Unusual inheritance of evolutionarily-related double-stranded RNAs in interspecific hybrid between rice plants Oryza sativa and Oryza rufipogon

Endogenous, 14 kb double-stranded RNAs (dsRNAs) have been found in two ecospecies of cultivated rice (temperate japonica rice and tropical japonica rice, Oryza sativa L.) and in wild rice (O. rufipogon, an ancestor of O. sativa). A comparison of the nucleotide and deduced amino acid sequences of the core regions of the RNA-dependent RNA polymerase domains found in these three dsRNAs suggested that these dsRNAs probably evolved independently within each host plant from a common ancestor. These dsRNAs were introduced into F1 hybrids by crossing cultivated rice and wild rice. Unusual cytoplasmic inheritance of these dsRNAs was observed in some F1 hybrids; the evolutionarily related dsRNAs were incompatible for each other, and the resident dsRNA of an egg cell from cultivated rice was excluded by the incoming dsRNA of a pollen cell from wild rice. Coexisting dsRNAs in the F1 hybrids segregated away from each other in the F2 plants. However, the total amount of these dsRNAs in the host cells remained constant (ca. 100 copies/cell). The stringent regulation of the dsRNA copy number may be responsible for their unusual inheritance.     

Double-stranded RNA replicons associated with chloroplasts of a green alga,Bryopsis cinicola

Double-stranded RNAs (dsRNAs) associated with chloroplasts and mitochondria have been found in the coenocytic green alga Bryopsis cinicola. In this study we report molecular properties of the four chloroplast-associated dsRNAs (BDRC1 to BDRC4) The longest dsRNA molecule (BDRC1) was sequenced entirely (1959 bp) and a single large ORF of 1722 bp was found within it. Database searches revealed similarities between the deduced amino acid sequence of this ORF and RNA-dependent RNA polymerase (RdRp) sequences from several RNA viruses. The most similar sequence in the database was the RdRp of beet cryptic virus 3. Phylogenetic analysis revealed that the RdRp-like sequence of BDRC1 can be placed in the Partitiviridae clade. To detect autonomous replication of these dsRNAs, RdRp assays were carried out with actinomycin D, which is an inhibitor of DNA-dependent RNA synthesis. Incorporation of [-³²P]UTP was detected specifically in the chloroplast and mitochondrial dsRNAs, indicating that both the chloroplast dsRNAs (BDRCs) and the mitochondrial dsRNA (BDRM) of B. cinicola are RNA replicons. The green alga B. cinicola harbors different dsRNA replicons in its chloroplasts and mitochondria.     

Stringently and developmentally regulated levels of a cytoplasmic double-stranded RNA and its high-efficiency transmission via egg and pollen in rice

A very restricted amount of high-molecular-weight double-stranded RNA (dsRNA) has been found in healthy japonica rice plants. We discriminated dsRNA-carrying rice plants from noncarriers. The endogenous dsRNA was localized in the cytoplasm (about 100 copies per cell) and was transmissible to progeny plants by mating. In crosses between carriers and noncarriers, the RNA was transmitted efficiently to F1 plants via both egg and pollen. The rice dsRNA was maintained at an almost constant level by host plant cells from generation to generation. The high-efficiency transmission of the endogenous dsRNA to progeny plants appears to depend on the autonomously controlled replication of the dsRNA localized in cytoplasmic vesicles. However, an increase in copy number (about 10-fold) of the dsRNA was observed during the suspension culture of host cells. The number of copies of dsRNA returned to the original low value in regenerated plants, suggesting that the copy number is stringently and developmentally regulated in rice cells.     

Enigmatic double-stranded RNA in Japonica rice

We have found a linear, 16 kb, double-stranded RNA (dsRNA) in symptomless Japonica rice (Oryza sativa L.) that is not found in Indica rice (Oryza sativa L.). The dsRNA was detected in every tissue and at every developmental stage, and its copy number was approximately constant (about 20 copies/cell). Double-stranded RNA was also detected in two strains of Oryza rufipogon (an ancestor of O. sativa). Hybridization experiments indicated that the dsRNA of O. rufipogon was homologous but not identical to that of O. sativa. The sequence of about 13.2 kb of the dsRNA was determined and two open reading frames (ORFs) were found. The larger ORF (ORF B) was more than 12 351 nucleotides long and encoded more than 4 117 amino acid residues.     

Double-stranded RNA and male sterility in rice

Summary Double-stranded RNA (dsRNA) was isolated from rice Oryza sativa ssp. japonica, but not from other subspecies. The dsRNA has been found in all of the examined cytoplasmic male-sterile (CMS) lines of BT (Chinsurah Boro II)-type rice, but was not detected in their companionate maintainer lines. It is uniquely and positivley correlated with the CMS trait in BT-type rice. Recently, the dsRNA was also found in a nuclear malesterile (NMS) rice, Nongken 58s, but was not found in its normal Nongken 58. The molecular weight of this dsRNA was estimated to be about 18 kb. Electron microscopic analysis reveals that it is linear snapped. The double strandedness of the RNA molecules was characterized by CF-11 cellulose column chromatography and nuclease treatments. It bound to CF-11 cellulose in the presence of 15% ethanol. It was sensitive to RNase A at low salt concentrations, but insensitive to DNase I, SI nuclease, and RNase A at high salt concentrations. The dsRNA was detected in both mitochondrial and cytoplasmic fractions. Dot-blot hybridization reveals that there is no sequence homology between this dsRNA and mtDNA, but there is homology between this dsRNA and nuclear genomic DNA. We have not been able to transmit this dsRNA to fertile rice.     

Association of particles that contain double-stranded RNAs with algal chloroplasts and mitochondria

Linear dsRNAs (double-stranded RNAs) belonging to several distinct size classes were found to be localized in chloroplasts and mitochondria of Bryopsis spp., raising the possibility that these dsRNAs are prokaryotic in nature. The algal cytosol and nuclei did not contain dsRNAs. The amount of the dsRNAs in the organelles appeared constant, and there were about 500 copies per chloroplast. The four major dsRNAs from Bryopsis chloroplasts were about 2 kbp (kilobase pairs) in length and originated from discrete isometric particles of about 25 nm diameter. These virus-like particles were purified by CsCl density gradient centrifugation after extraction from isolated chloroplasts with chloroformbutanol and subsequent precipitation with polyethylene glycol. They had a buoyant density of about 1.40 g · cm^–3 and contained four major and three minor proteins. Mitochondrial dsRNAs were about 4.5 kbp in length and formed less-stable particles of about 40 nm in diameter with a buoyant density of 1.47 g · cm^–3. Some observations support the hypothesis that vertical transmission of the protein-coated, non-infectious dsRNAs occurs within cell organelles. Double-stranded RNAs of various sizes were found in most green, red, and brown algae. The characteristics of the algal dsRNAs are compared with those of dsRNAs from higher plants and the biological significance of the dsRNAs in cell organelles is discussed.Abbreviations dsRNA double-stranded RNA - kbp kilobase pairs - SDS sodium dodecyl sulfate - SSC 0.15 M NaCl 0.015 M sodium citrate - PAGE polyacrylamide gel electrophoresis The authors would like to express their gratitude to Dr. T. Natsuaki, Utsunomiya University, and Dr. D. Hosokawa, Tokyo University of Agriculture and Technology, for their helpful suggestions throughout this research. They are also much indebted to Dr. B. Wang, Institute of Genetics, Academia Sinica, Beijing, PRC, for his suggestions on rice dsRNA, and to Dr. T. Kohbara, Senshu University, on Bryopsis cells. Sincere thanks are also due to Dr. T. Misonou, Yamanashi University, and Dr. K. Masuda, Akita Prefectural College of Agriculture, for supplying plant materials; to Dr. N. Sonoki, Azabu University, for nucleotide analysis of dsRNAs; and to Y. Koshino for technical assistance. This research was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

Double-stranded RNA in rice: A novel RNA replicon in plants

The entire sequence of 13952 nucleotides of a plasmid-like, double-stranded RNA (dsRNA) from rice was assembled from more than 50 independent cDNA clones. The 5 non-coding region of the coding (sense) strand spans over 166 nucleotides, followed by one long open reading frame (ORF) of 13716 nucleotides that encodes a large putative polyprotein of 4572 amino acid residues, and by a 70-nucleotide 3 noncoding region. This ORF is apparently the longest reported to date in the plant kingdom. Amino acid sequence comparisons revealed that the large putative polyprotein includes an RNA helicase-like domain and an RNA-dependent RNA polymerase (replicase)-like domain. Comparisons of the amino acid sequences of these two domains and of the entire genetic organization of the rice dsRNA with those found in potyviruses and the CHV1-713 dsRNA of chestnut blight fungus suggest that the rice dsRNA is located evolutionarily between potyviruses and the CHV1-713 dsRNA. This plasmid-like dsRNA in rice seems to constitute a novel RNA replicon in plants.     

Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of a mRNA containing the same sequence. The 21–23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that dsRNA specifically suppresses the expression of HIV-1 genes. To study dsRNA-mediated gene interference in HIV-1-infected cells, we have designed six long dsRNAs containing the HIV-1 gag and env genes. HIV-1 replication was totally suppressed in a sequence-specific manner by the dsRNAs in HIV-1-infected cells. Especially, E2 dsRNA containing the major CD4-binding domain sequence of gp120, as the target of the HIV-1 env gene, dramatically inhibited the expression of the HIV-1 p24 antigen in PBMCs for a relatively long time. The dsRNA interference method seems to be a promising new strategy for anti-HIV-1 gene therapeutics.     

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Abstract Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double‐stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome‐integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.     

Transmissibility of viral double-stranded RNA between strains of the violet root rot fungus <Emphasis Type="Italic">Helicobasidium mompa </Emphasis>and the potential for viral dsRNA infection to this fungus using monokaryotic strains

 To examine the potential of a method of double-stranded (ds) RNA infection to Helicobasidium mompa, the transmissibility of dsRNA between strains of this fungus was investigated. Strain V70 was used as a dsRNA donor. The dsRNA recipients were six strains that were mycelially incompatible with V70, plus two monokaryotic strains. Random amplified polymorphic DNA analysis suggested that the mycelially incompatible strains were genetically different mutually; however, the analysis also suggested that V70 was genetically homogeneous with the two monokaryotic strains. When V70 was paired with either of the mycelially incompatible strains, the dsRNAs did not transmit to the recipients at all. When V70 was paired with the two monokaryotic strains, the dsRNAs were transmitted to the monokaryotic strains. The two monokaryotic strains, which had acquired dsRNAs from V70 in the previous experiment, were used as new dsRNA donors in a next experiment so that we could investigate dsRNA transmission from these monokaryotic strains to the six strains used in the previous experiment. One of the monokaryotic strains permitted dsRNA transmission to two of the six recipients. We conclude that we can infect genetically different strains of H. mompa with dsRNA using the monokaryotic strains. Received: December 12, 2002 / Accepted: January 27, 2003 Acknowledgments This research was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences. The authors are grateful to Dr. Tadanori Aimi of Tottori University for helpful discussion. Correspondence to:K. Suzaki     

Molecular characterization of a single mitochondria-associated double-stranded RNA in the green alga Bryopsis

Mitochondria from the green alga Bryopsis sp. very often contained a 4.5 kb double-stranded RNA (dsRNA) at a defined level. Complementary DNA probes derived from the mitochondrial dsRNA hybridized with none of the algal chloroplast dsRNAs of 1.7 to 2.2 kb, but did hybridize with a similar-sized dsRNA among several dsRNAs from the mitochondria of B. maxima. Sequence analysis of the mitochondrial dsRNA from Bryopsis sp. revealed only two large, overlapping, open reading frames (ORFs) on one strand if UGA was taken as a non-termination codon, suggesting the independent phylogenetic evolution of the mitochondrial dsRNA. Consensus sequence for RNA-dependent RNA polymerase was found within the longer ORF (2472 bp) of the dsRNA. The overlapping 52 bp of the ORFs in different reading frames is suggestive of the occurrence of a -1 ribosomal frameshift in the mitochondrial translation system. The observed simple genetic structures suggest that the algal mitochondrial dsRNA might be deficient in a gene for movement from cell to cell in host plants and, hence, has a plasmid-like nature that is distinct from that of infectious plant viruses. The nature and origin of the endogenous dsRNAs of various sizes and their relationships are discussed.     

Systemic RNAi in western corn rootworm,Diabrotica virgifera virgifera,does not involve transitive pathways

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double‐stranded RNA (dsRNA). RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA‐dependent RNA polymerase (RdRP) activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi‐mediated knockdown of Dv v‐ATPase C mRNA throughout the WCR gut and other tissues using high‐sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v‐ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.     

Chitosan nanoparticles help double-stranded RNA escape from endosomes and improve RNA interference in the fall armyworm,Spodoptera frugiperda

RNA interference (RNAi) is a promising technology for the development of next-generation insect pest control products. Though RNAi is efficient and systemic in coleopteran insects, it is inefficient and variable in lepidopteron insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda by conjugating double-stranded RNA (dsRNA) with biodegradable chitosan (Chi). dsRNA conjugated with chitosan was protected from degradation by endonucleases present in Sf9 cell-conditioned medium, hemolymph, and midgut lumen contents collected from the FAW larvae. Chi–dsRNA complexes showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing chitosan formulated dsRNA in Sf9 cells and the tissues induced a significant knockdown of endogenous genes. Chi–dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation, and mortality. Processing of dsRNA into small interfering RNA was detected with chitosan-conjugated ³²P-UTP-labeled ds green fluorescent protein in Sf9 cells and FAW larval tissues. Overall, these data suggest that dsRNA conjugated with chitosan helps dsRNA escape from the endosomes and improves RNAi efficiency in FAW cells and tissues.     

Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos

BACKGROUND: RNA interference (RNAi) is a phenomenon in which introduced double-stranded RNAs (dsRNAs) silence gene expression through specific degradation of their cognate mRNAs. Recent analyses in vitro suggest that dsRNAs may be copied, or converted, into 21-23 nucleotide (nt) guide RNAs that direct the nucleases responsible for RNAi to their homologous mRNA targets. Such small RNAs are also associated with gene silencing in plants. RESULTS: We developed a quantitative single-embryo assay to examine the mechanism of RNAi in vivo. We found that dsRNA rapidly induced mRNA degradation. A fraction of dsRNAs were converted into 21-23 nt RNAs, and their time of appearance and persistence correlated precisely with inhibition of expression. The strength of RNAi increased disproportionately with increasing dsRNA length, but an 80bp dsRNA was capable of effective gene silencing. RNAi was saturated at low dsRNA concentration and inhibited by excess unrelated dsRNA. The antisense strand of the dsRNA determined target specificity, and excess complementary sense or antisense single-stranded RNAs (ssRNAs) competed with the RNAi reaction. CONCLUSIONS: Processed dsRNAs can act directly to mediate RNAi, with the antisense strand determining mRNA target specificity. The involvement of 21-23 nt RNAs is supported by the kinetics of the processing reaction and the observed size dependence. RNAi depends on a limiting factor, possibly the nuclease that generates the 21-23 mer species. The active moiety appears to contain both sense and antisense RNA strands.     

Short double-stranded RNAs with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as RIG-I decoys

Arenavirus RNA genomes are initiated by a "prime and realign" mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to blunt-ended (5' ppp)dsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes. We examined the ability of various dsRNAs to activate the RIG-I ATPase (presumably a measure of helicase translocation on dsRNA) relative to their ability to induce IFN. We found that there is no simple relationship between these two properties, as if RIG-I can translocate on short dsRNAs without inducing IFN. Moreover, we found that (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide can in fact competitively inhibit the ability of blunt-ended (5' ppp)dsRNAs to induce IFN when co-transfected into cells and that this inhibition is strongly dependent on the presence of the 5' ppp. In contrast, (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide does not inhibit poly(I-C)-induced IFN activation, which is independent of the presence of a 5' ppp group.     

Molecular characterisation of an endornavirus from Rhizoctonia solani AG-3PT infecting potato

Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is a soil-borne plant pathogenic fungus that has a broad host range, including potato. In this study, the double-stranded RNA (dsRNA) profiles were defined for 39 Rhizoctonia solani isolates representative of two different anastomosis groups (AGs) associated with black scurf of potato in New Zealand. A large dsRNA of c. 12 kb–18 kb was detected in each of the isolates, regardless of AG or virulence on potato. Characterisation of the large dsRNA from R. solani AG-3PT isolate RS002, using random amplification of total dsRNA and analyses of overlapping cDNA sequences, resulted in the assembly of a consensus sequence of 14 694 nt. A single, large open reading frame was identified on the positive strand of the assembled sequence encoding a putative polypeptide of at least 4893 amino acids, with a predicted molecular mass of 555.6 kDa. Conserved domains within this polypeptide included those for a viral methyltransferase, a viral RNA helicase 1 and an RNA-dependent RNA polymerase. The domains and their sequential organisation revealed the polyprotein was very similar to those encoded by dsRNA viruses of the genus Endornavirus, in the family Endornaviridae. This is the first report of an endornavirus in R. solani, and thus the putative virus is herein named Rhizoctonia solani endornavirus - RS002 (RsEV-RS002). Partial characterisation of the large dsRNAs in five additional AG-3PT isolates of R. solani also identified them as probable endornaviruses, suggesting this family of viruses is widespread in R. solani infecting potato. The ubiquitous nature of endornaviruses in this plant pathogen implies they may have an important, but yet uncharacterised, role in R. solani.