Biophysical studies on double-stranded RNA from turnip yellow mosaic virus-infected plants

Summary Double-stranded RNA was isolated in mg quantities from TYMV-infected cabbage plants by a modified phenol procedure. Chromatography of the RNA on methylated albumin and hydroxyapatite is described. The base composition (A=21.3; U=21.2; G=28.8; C=28.7) was in good agreement with the values expected for a double-stranded RNA consisting of TYMV RNA and a strand complementary to it. The buoyant density of the RNA in Cs₂SO₄ was 1.617 g/cm³. Single-stranded TYMV RNA banded at 1.642 g/cm³ in Cs₂SO₄. The RNA sedimented in the analytical ultracentrifuge with an average sedimentation coefficient of 10–11 s. Absorbance as a function of temperature was determined in several different media. The absorbance-temperature profiles were typical of those expected for double-stranded RNA. Denatured RNA was examined by equilibrium density gradient centrifugation.     

Further studies on ultrastructure of plants infected withPetunia ringspot virus

Summary New methods of fixation and embedding have revealed in plants infected withPetunia ringspot some structural features not described before. These include the X-bodies, 80 per cent of which are formed by tubular elements which are responsible for the positive staining specific for Golgi apparatus. The tubular elements are morphologically similar to agranular endoplasmic reticulum as described in some animal cells. The rest of the inclusion is formed by normal cytoplasmic elements in which are embedded long rod-shaped tubules 600 Å wide and more than 7,000 Å long, cross sections of which are formed by 10 subunits. These subunits are arranged in a helical way to form the large tubules. These subunits are probably the actual virus particles, which would be icosahedral and would form tubular crystals. Icosahedral virus particles would also form true crystalline inclusions.It is not known what the role of the agranular endoplasmic reticulum and of some dense osmiophilic bodies found in it may be in the multiplication of the virus. However, these components induced by the virus infection probably result in the manufacture of some proteins or other substances necessary for virus multiplication.     

Parallels among positive-strand RNA viruses, reverse-transcribing viruses and double-stranded RNA viruses

Viruses are divided into seven classes on the basis of differing strategies for storing and replicating their genomes through RNA and/or DNA intermediates. Despite major differences among these classes, recent results reveal that the non-virion, intracellular RNA-replication complexes of some positive-strand RNA viruses share parallels with the structure, assembly and function of the replicative cores of extracellular virions of reverse-transcribing viruses and double-stranded RNA viruses. Therefore, at least four of seven principal virus classes share several underlying features in genome replication and might have emerged from common ancestors. This has implications for virus function, evolution and control.     

Further studies on resistance to Leptosphaeria coniothyrium in the red raspberry and related species

Rubus pileatus and its F₁ hybrids with raspberry (R. idaeus) were resistant to cane blight (Leptosphaeriu conioth-yriurn), but little resistance was obtained in subsequent backcross generations apart from a hybrid identified in the second backcross. Two hybrids from backcrossing R. coreanus to raspberry also showed resistance. R. pileatus and its F, hybrids produced hard growth, unlike that of raspberries, which may have been associated with a form of resistance that could not easily be transferred to commercial raspberry cultivars. Some of the genotypes used as parents showed intermediate levels of resistance and it is possible that the highly resistant genotype identified in the second backcross arose from a recombination of genes for resistance. Plants with pubescent canes (gene H) were up to 20% more resistant to mycelial inoculation than those with non-pubescent canes (gene h), and the percentage of machine-harvester inflicted wounds with disease symptoms that resulted from natural infection was also less in genotypes with pubescent canes. Many genotypes with intermediate levels of resistance suffered only limited damage from mycelial inoculation and so there are good prospects for breeding cultivars with an effective resistance, despite the limited value of R. pileatus as a donor species.     

A closely related group of RNA-dependent RNA polymerases from double-stranded RNA viruses.

Probably one of the first proteinaceous enzymes was an RNA-dependent RNA polymerase (RDRP). Although there are several conserved motifs present in the RDRPs of most positive and double-stranded RNA (dsRNA) viruses, the RDRPs of the dsRNA viruses show no detectable sequence similarity outside the conserved motifs. There is now, however, a group of dsRNA viruses of lower eucaryotes whose RDRPs are detectably similar. The origin of this sequence similarity appears to be common descent from one or more noninfectious viruses of a progenitor cell, an origin that predates the differentiation of protozoans and fungi. The cause of this preservation of sequence appears to be constraints placed on the RDRP by the life-style of these viruses--the maintenance of a stable, persistent, noninfectious state.     

The effect of resistance to Amphorophora rubi in raspberry (Rubus idaeus) on the spread of aphid-borne viruses

The effectiveness of resistance to the aphid Amphorophora rubi in restricting the spread of aphid-borne viruses was assessed in a field experiment using six genotypes of red raspberry. In one block of the experiment, the genotypes alternated with rows of virus-infected Mailing Jewel raspberry, and in the other they alternated with virus-free Mailing Jewel. During 4 years, the numbers of A. rubi and the amount of 52V virus spread in the two blocks were similar, suggesting that this virus was mostly introduced from outside the plots. Lloyd George and Mailing Jewel raspberry became heavily infested with A. rubi and were rapidly infected with raspberry leaf mottle, raspberry leaf spot and 52V viruses. Glen Clova and Norfolk Giant raspberry, which contain minor genes for resistance to A. rubi, were infested with fewer A. rubi and virus spread more slowly in these cultivars. A. rubi were rare on Mailing Orion and an East Mailing raspberry selection (888/49) which have genes A₁ and A₁₀ respectively for resistance to A.rubi, and these plants remained largely free of virus. The role of minor and major gene resistance to A. rubi in restricting virus spread is discussed. A few Macrosiphum euphorbiae and Myzus ornatus were recorded on several of the raspberry genotypes.     

Epigenetic modification of plants with systemic RNA viruses

Knowledge of gene function is critical to the development of new plant traits for improved agricultural and industrial applications. Viral expression vectors offer a rapid and proven method to provide epigenetic expression of foreign sequences throughout infected plants. Expression of these sequences from viral vectors can lead to gain- or loss-of-function phenotypes, allowing gene function to be determined by phenotypic or biochemical effects in the infected plant. Tobacco mosaic virus and barley stripe mosaic virus expression vectors have been developed to express foreign gene sequences in dicotyledonous and monocotyledonous hosts, respectively. Large-scale application of both viral vector systems for gene function discovery in Nicotiana and barley hosts resulted in high infection rates and produced distinctive visual phenotypes in approximately 5% of transfected plants. Novel genes expressing potential herbicide target proteins in addition to genes promoting stem elongation, leaf development and apical dominance were identified in the large-scale screening. This report illustrates the adaptability of viral vectors for gene function discovery in higher plants.     

Rubus host range of rubus yellow net virus and its involvement with other aphid-borne latent viruses in inducing raspberry veinbanding mosaic disease

After graft inoculation with rubus yellow net virus (RYNV), 12 of 34 Rubus species and cultivars developed noticeable symptoms. R. macraei developed the most conspicuous symptoms and is recommended as an improved indicator plant. In attempts to determine the cause of raspberry veinbanding mosaic, a disease in which RYNV is involved, several European and North American red raspberry cvs were graft-inoculated with RYNV and three other aphid-borne viruses, black raspberry necrosis (BRNV), raspberry leaf mottle (RLMV) and raspberry leaf spot, singly and in all combinations. In periods of up to 4 yr, classical veinbanding mosaic symptoms developed in sensitive cvs only when they contained both RYNV and RLMV. These symptoms were intensified in plants co-infected with additional viruses. Veinbanding mosaic disease did not develop in any of 11 cvs infected with RYNV + BRNV, the combination of viruses previously assumed to be responsible for this disease in Britain and North America.     

The roles of ascorbic acid and glutathione in symptom alleviation to SA-deficient plants infected with RNA viruses

Salicylic acid (SA) is required for plant systemic acquired resistance (SAR) to viruses. However, SA-deficient plants adapt to RNA virus infections better, which show a lighter symptom and have less reactive oxygen species (ROS) accumulation. The virus replication levels are higher in the SA-deficient plants during the first 10?days, but lower than the wild-type seedlings after 20?dpi. The higher level of glutathione and ascorbic acid (AsA) in SA-deficient plants may contribute to their alleviated symptoms. Solo virus-control method for mortal viruses results in necrosis and chlorosis, no matter what level of virus RNAs would accumulate. Contrastingly, early and high-dose AsA treatment alleviates the symptom, and eventually inhibits virus replication after 20?days. ROS eliminators could not imitate the effect of AsA, and could neither alleviate symptom nor inhibit virus replication. It suggests that both symptom alleviation and virus replication control should be considered for plant virus cures.     

RNA沉默与植物病毒

植物中RNA沉默（RNAsilencing)亦称为转录后基因沉默（PTGS）或共抑制,是植物抵抗外来核酸（转座子、转基因或病毒）入侵,并保护自身基因组完整性的一种防御机制。RNA沉默是近十年来发现的植物界中普遍存在的现象,已成为植物分子生物学领域的一个新的研究方向。对RNA沉默特点和机制的研究表明,植物病毒与（转基因）植物内发生的RNA沉默有着密切的联系,作者从病毒对RNA沉默的诱导、抑制、防御等方面,简述了RNA沉默与病毒的关系。并对病毒载体所诱导的RNA沉默在植物发育和基因组功能分析等方面的应用价值进行了讨论。     

A plasmid-based reverse genetics system for animal double-stranded RNA viruses

Mammalian orthoreoviruses (reoviruses) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis. Reoviruses infect respiratory and intestinal epithelium and disseminate systemically in newborn animals. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of the Reoviridae family of dsRNA viruses. We report the generation of viable reovirus following plasmid transfection of murine L929 (L) cells using a strategy free of helper virus and independent of selection. We used the reovirus reverse genetics system to introduce mutations into viral capsid proteins sigma1 and sigma3 and to rescue a virus that expresses a green fluorescent protein (GFP) transgene, thus demonstrating the tractability of this technology. The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector.     

Further studies on breeding for resistance to Botrytis cinerea in red raspberry canes

A high level of resistance to cane botrytis caused by Botrytis cinerea was transferred from Rubus pileatus to the red raspberry through three generations of backcrossing. The strength of the resistance showed little diminution through these generations and it was therefore thought that a major gene for resistance had been transferred. But discontinuity in the resistance levels observed was detected in plants of only one group of the progenies and so evidence for a major gene is lacking. The results showed the considerable influence of gene H, which confers pubescence, and emphasised that resistance must be separately assessed for pubescent and non-pubescent segregates.     

Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes.

Infectious pancreatic necrosis virus of fish, infectious bursal disease virus of chickens, Tellina virus and oyster virus of bivalve molluscs, and drosophila X virus of Drosophila melanogaster are naked icosahedral viruses with an electron microscopic diameter of 58 to 60 nm. The genome of each of these viruses consists of two segments of double-stranded RNA (molecular weight range between 2.6 x 10(6) and 2.2 x 10(6), and the virion, capsid proteins fall into three size class categories (large, medium, and small; ranging from 100,000 to 27,000) as determined by polyacrylamide slab gel electrophoresis. The hydrodynamic properties of the five viruses are similar as determined by analytical ultracentrifugation and laser quasi-elastic, light-scattering spectroscopy. The calculated particle weights range between 55 x 10(6) and 81 x 10(6). Tryptic peptide comparisons of 125I-labeled virion proteins showed that five viruses are different from each other, although there was considerable overlap in the peptide maps of the three aquatic viruses, indicting a degree of relatedness. Cross-neutralization tests indicated that drosophila X, infectious pancreatic necrosis, and infectious bursal disease viruses were different from each other and from oyster and Tellina viruses. The same test showed oyster and Tellina viruses to be related. The biochemical and biophysical properties of the five viruses cannt be included in the family Reoviridae or in any of the present virus genera.     

Superkiller yeast strain contains additional species of double-stranded RNA

A superkiller yeast strain, T158C, was found to contain four new species of ds RNA designated P3, P4, P5, and P6. The new components were found to have molecular weights of 880,000 daltons, 620,000 daltons, 500,000 daltons, and 330,000 daltons, respectively. The new species were shown to be double-stranded RNA by their resistance to pancreatic RNAse (E.C.3.1.4.22) in high salt and their sensitivity in low salt. Curing of the killer character by growth at high temperature resulted in the loss of P3–6 as well as M ds RNA. Since T158C is a superkiller, these new molecules may play an important role in the expression of the killer character.     

Molecular characterisation of subgenomic single-stranded and double-stranded DNA forms isolated from plants infected with tomato golden mosaic virus.

A subgenomic single-stranded DNA present in particles of the geminivirus, tomato golden mosaic virus, has been shown by electron microscope heteroduplex mapping and Southern hybridisation analysis to consist of circular molecules, ca. 1.2 kb in size, derived from the smaller of the two genomic DNA components, DNA B, by deletion of open reading frame (ORF) BR1 and the C-terminal portion of ORF BL1. A covalently closed circular, supercoiled, double-stranded form of the subgenomic DNA has been isolated from virus-infected plants and cloned into pEMBL9. Analysis of the sequence of 22 clones across the deletion boundaries revealed only four different deletion boundaries, derived from four different left hand borders and three different right hand borders. Each border was within a region of 11 nucleotides and gave rise to a narrow size range (1248-1261 nucleotides) for the population of 22 subgenomic DNAs. However apparently smaller subgenomic DNAs were sometimes formed when plants were inoculated with cloned subgenomic DNA, or a construct derived from a subgenomic DNA in which a neomycin phosphotransferase gene had been inserted, together with the genomic DNA components. Mechanisms to account for the size, specificity and formation of the subgenomic DNA are discussed.