Double Membrane Bodies Research Articles

Characterization of a Novel Emaravirus Affecting Ash Species (Fraxinus spp.) in Europe

We identified a novel virus in diseased European ash (Fraxinus excelsior) and manna ash (F. ornus) trees exhibiting chlorotic ringspots, mottle and leaf deformation such as curling and shoestring symptoms. High-throughput sequencing (HTS, Illumina RNASeq) of total RNA isolated from diseased leaf material in combination with RT-PCR-based amplification techniques and Sanger sequencing determined five complete genome segments, each encoding a single open reading frame. Sequence analyses of RNA1–RNA5 revealed a genome organization typical for emaraviruses, i.e., (i) conserved and complementary terminal 5′ and 3′ termini of each genome segment (ii) proteins showing significant homologies to the RNA-dependent RNA polymerase (RdRP) encoded by RNA1, the glycoprotein precursor (GPP) encoded by RNA2, the viral nucleocapsid protein (N, RNA3), the movement protein (MP, RNA4), and a protein of 26 kDA (P26, RNA5) highly similar to proteins of unknown function encoded by other emaraviruses. Furthermore, we identified spherical particles (double-membrane bodies, DMB) of different sizes (70–80 nm in diameter) which are typical for emaraviruses exclusively in virus-infected leaf tissue exhibiting mottle and leaf deformation. Sequence comparison and phylogenetic analyses confirmed the identified novel virus as a new member of the genus Emaravirus. We established a species-specific RT-PCR detection protocol and could associate the observed disease symptoms with the infection of the novel emaravirus in F. excelsior and F. ornus. Therefore, we propose the name ash shoestring-associated emaravirus (ASaV). Investigation of ASaV-infected sample trees originating from different locations in Switzerland, Germany, Italy and Sweden provided a wide geographical distribution of the virus in affected ash species. To our knowledge, this is the first confirmation of an emaravirus affecting ash tree species with shoestring symptoms of leaves in Europe.

Autophagy-Like Cell Death Regulates Hydrogen Peroxide and Calcium Ion Distribution in Xa3/Xa26-Mediated Resistance to Xanthomonas oryzae pv. oryzae.

The broad-spectrum and durable resistance gene Xa3/Xa26 against Xanthomonas oryzae pv. oryzae (Xoo) has been widely exploited in rice production in China. But the cytological features of the Xa3/Xa26-mediated resistance reaction have been rarely reported. This study reveals the cytological characteristics of the Xa3/Xa26-mediated resistance reaction against Xoo to uncover the functions of hypersensitive response programmed cell death (HR-PCD) in rice. Autophagy-like cell death, which was characterized by double-membrane bodies appearance in xylem parenchyma cell and mesophyll cell, was inhibited by autophagy inhibitor 3-methyladenin (3-MA). The autophagy-related genes were induced to reach a high level in resistance reaction. The hydrogen peroxide (H2O2) maintained a low concentration on the plasma membrane. The calcium ions localized on the apoplast were transferred into the vacuole. The autophagy inhibitor (3-MA) impaired Xa3/Xa26-mediated resistance by promoting the accumulation of H2O2, and inhibited the transfer of extracellular calcium ions into the vacuole in the xylem parenchyma cells and mesophyll cells. Therefore, the HR-PCD belongs to autophagy-like cell death in the Xa3/Xa26-mediated resistance reaction. These results suggest that the autophagy-like cell death participates in the Xa3/Xa26-mediated resistance by negatively regulating H2O2 accumulation, in order to abolish oxidative stress and possibly activate calcium ion signals in xylem parenchyma cells of the rice leaf.

Characterization of Ti Ringspot-Associated Virus, a Novel Emaravirus Associated with an Emerging Ringspot Disease of Cordyline fruticosa.

Ti ringspot is an emerging foliar disease of the ti plant (Cordyline fruticosa) in Hawaii that is quickly spreading throughout the islands. Symptoms include small chlorotic ringspots on leaves that often coalesce to form larger lesions. Although several virus species have been discovered in symptomatic plants, none have been associated with these symptoms. Here, we report and characterize a novel virus closely associated with ti ringspot symptoms in Hawaii. The presence of double membrane bodies approximately 85 nm in diameter in symptomatic cells and sequence analyses of five genomic RNA segments obtained by high-throughput sequencing indicate that this virus is most closely related to members of the plant virus genus Emaravirus. Phylogenetic and sequence homology analyses place this virus on a distinct clade within the Emaravirus genus along with High Plains wheat mosaic emaravirus, blue palo verde broom virus, and Raspberry leaf blotch emaravirus. Sequence identity values with taxonomically relevant proteins indicate that this represents a new virus species, which we are tentatively naming ti ringspot-associated virus (TiRSaV). TiRSaV-specific reverse transcription PCR assays detected the virus in several experimental herbaceous host species following mechanical inoculation. TiRSaV was also detected in eriophyid mites collected from symptomatic ti plants, which may represent a putative arthropod vector of the virus.

Micropropagation of virus-free plants of Saudi fig (Ficus carica L.) and their identification through enzyme-linked immunosorbent assay methods

Viral infection is one of the most serious biotic stresses, which disturbs the growth and productivity of many horticultural crops, including that of fig (Ficus carica L.). The production of plants free of viruses, such as fig mosaic virus (FMV), has become a priority in many plant breeding programs. In this study, leaves from plants of two fig cultivars, Kodato and Dattora, infected with FMV were collected from both Mecca and Al-Taif, Saudi Arabia. Transmission electron microscopy of ultrathin leaf sections showed double membrane bodies, characteristic of FMV particles, only in the mesophyll cells of infected samples. Protein analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a protein band with a molecular weight of 35 kDa, which corresponded to the viral coat protein; and FMV was confirmed by Western blot and enzyme-linked immunosorbent assay (ELISA) tests. To obtain virus-free plants, apical shoot culture was applied. A comparison of various artificial media with different concentrations of growth regulators was evaluated to optimize shoot formation, shoot multiplication, and root formation, and was followed by plant acclimation ex vitro. Direct ELISA analysis of shoots micropropagated from meristem tip explants indicated that there were virus-free shoots, when compared to infected plants (positive control), while there were no significant differences between these explants and healthy samples (negative control). This study demonstrated that in vitro micropropagation of Saudi F. carica infected with FMV virus led to the successful elimination of the virus.

Rose rosette virus: An emerging pathogen of garden roses in India

Rose rosette virus (RRV) was characterized as a new member of the virus genus Emaravirus, having a seven single-stranded negative-sense RNA genome. In this communication, partial RdRp gene of RRV has been characterized from infected roses through reverse transcription-PCR using RRV specific primers. The virus was detected in all twenty symptomatic plants tested from the north-eastern region of India and the partial RdRp genes of two isolates were sequenced. Electron microscopy showed the presence of double membrane bodies in all the infected leaf tissues. The three dimensional structure of the RdRp gene of the two isolates was predicted through homology modelling. The partial putative protein of the Siliguri and Siliguri2 isolates of RRV contained a signature motif B (QGNLNMISS) of the Bunyaviridae-RdRp domain. Phylogenetic analysis of these two RRV isolates showed 95–98% sequence identity with other RRV isolates previously described worldwide. After 75 years since the first report, RRV is being reported as an emerging disease of garden roses in India.

DETECTION OF FIG MOSAIC VIRUS IN VIRULIFEROUS ERIOPHYID MITE ACERIA FICUS

Fig leaves showing typical fig mosaic symptoms on cv. Bursa siyahi (donor plant) were cut under a stereo microscope into small pieces each hosting 10 putatively viruliferous eriophyid mites (Aceria ficus Cotte) and placed directly on the top leaves of healthy Cucumis sativus, Chenopodium quinoa. C. amaranticolor, Nicotiana occidentalis, Catharanthus roseus, Fraxinus excelsior plants, and fig seedlings. Donor and test plants were analyzed by electron microscopy, RT-PCR and sequencing, whereas the mites (ErMs) underwent molecular assays using Fig mosaic virus (FMV)-specific primers. Mite-infested leaves of fig seedlings and C. roseus showed small yellowish spots after 10 days and 6 weeks, respectively, whereas no symptoms were observed in other test or control plants for three months. Electron microscopy observations showed the occurrence of double membrane bodies (DMBs) in the palisade cells of donor and mite-inoculated fig plants, but not in C. roseus. However, 302 bp RT-PCR products specific to FMV were amplified from donor and inoculated figs, C. roseus and ErMs. Nucleotide identity with the sequence of the FMV isolate in GenBank (accession No. AM941711.6) was 87%, 89% and 87% for donor plant (JQ708183), inoculated fig seedlings (JQ708184) and C. roseus (JQ408437, JQ408438), respectively. The sequences obtained from ErMs (JQ408432, JQ408436) showed 87% and 88% nucleotide identity with the reference FMV isolate, respectively. When dsRNA extracts were analyzed to confirm virus presence in inoculated periwinkles, a complex dsRNA profile was obtained, suggestive of infection by a multipartite virus or by multiple viruses. Sequence from RT-PCR amplicons of dsRNA (JX040436) showed 88% identity with those the reference FMV isolate (AM941716.1) and the donor plant (JQ708183). According to these results, Madagascar periwinkle (C. roseus) can be retained as a new experimental host for FMV and A. ficus appears to be able to transmit FMV from fig to periwinkle plants.

Comparison by Sequence-Based and Electron Microscopic Analyses of Fig mosaic virus Isolates Obtained from Field and Experimentally Inoculated Fig Plants.

Fig mosaic disease (FMD) and the fig mite, Aceria ficus, are widespread in different fig growing provinces of Turkey. Fig trees (Ficus carica) cv. Bursa siyahı (D1) and an unknown seedling (D2) that showed typical FMD symptoms and was heavily infested by fig mites were used as donor plants for attempted mite transmissions to healthy fig seedlings. Transmission electron microscopy observations of donor plant samples prior to the transmission tests were performed and showed the presence of double membrane bodies (DMBs) in the palisade mesophyll cells. Electron microscopy of all experimentally inoculated fig seedlings showed the same bodies. This result reinforced the suggestion that an agent that elicits the production of DMBs in infected cells is involved in the etiology of FMD. Double-stranded (ds)RNA analyses were also performed from experimentally inoculated plants, and dsRNAs with sizes approximately 1.30 and 1.96 kb were obtained. Reverse transcription-polymerase chain reaction (RT-PCR) products of 468 and 298 bp specific to Fig mosaic virus (FMV) were amplified from both donor and experimentally inoculated plants. BLAST analyses of nucleotide sequences of these fragments showed 90% identity with FMV for the donor plant and 94 to 96% for experimentally inoculated plants. According to these results, FMV is present in both donor and experimentally inoculated plants in Turkey, and this virus is transmissible by A. ficus from fig plant to fig plant.

A multipartite single-stranded negative-sense RNA virus is the putative agent of fig mosaic disease

Several dsRNA bands (approx. 0.6-7 kbp in size) were recovered from tissues of mosaic-diseased fig seedlings which contained the enveloped round structures known as double membrane bodies (DMBs). blast analysis of a 4353 and a 1120 nt sequence from the two largest RNA segments showed homology with the polymerase and the putative glycoprotein precursor genes of negative-sense single-stranded RNA viruses of the family Bunyaviridae. Negative- and positive-sense riboprobes designed from both RNA segments hybridized to two bands of approximately 7 and 2.3 kbp in Northern blots of dsRNAs. Thus, these segments were identified as putative RNA-1 and RNA-2 of a novel virus for which the name fig mosaic virus (FMV) is proposed. Identity levels of predicted amino acids of the protein encoded by FMV RNA-1 with those of species of the family Bunyaviridae and European mountain ash ringspot-associated virus (EMERaV) were 28 and 54 %, respectively. RNA-2 showed 38 % identity at the amino acid level only with EMARaV. RNA-1 segment contained five conserved motifs (A-E) and an endonucleolytic centre of comparable genes of L RNA of bunyaviruses and EMARaV RNA-1. In a phylogenetic tree constructed with RdRp sequences, EMARaV grouped with FMV in a clade distinct from those of all bunyavirus genera. The consistent association of DMBs with mosaic symptoms and the results of molecular investigations strongly indicate that DMBs are particles of FMV, the aetiological agent of fig mosaic disease.

FIG MOSAIC IN MEXICO AND SOUTH AFRICA

Different patterns of chlorotic to yellowish mottling and deformation were observed in leaves of field-grown fig trees in Mexico and South Africa. Potted rooted cuttings from both sources grown under glasshouse conditions displayed chlorotic blotching, vein clearing and banding of the leaves. Electron microscope observations of thin-sectioned tissues from symptomatic leaves showed that parenchyma cells of both Mexican and South African samples contained the double-membrane bodies (DMBs) typically associated with fig mosaic disease (FMD). Also, accumulations of filamentous semi-rigid and of very flexuous virus-like particles were observed in parenchyma or phloem cells, respectively. RT-PCR assays of total nucleic acids extracted from leaf tissues of both sources using primers designed to amplify the HSP70 gene of the putative closteroviruses Fig leaf mottle-associated virus 1 (FLMaV-1) and Fig leaf mottle-associated virus 2 (FLMaV-2) gave a positive response only for FLMaV-1. DMBs have been observed previously in the symptomatic Mexican fig source. However, our results are the first records of FMD and associated DMBs in South Africa and of both FLMaV-1 and an unidentified filamentous semi-rigid virus in Mexico and South Africa.

Inhibition of VMAT-2 and DT-Diaphorase Induce Cell Death in a Substantia Nigra-Derived Cell LineAn Experimental Cell Model for Dopamine Toxicity Studies

We have induced intracellular dopamine oxidation to aminochrome in RCSN-3 cells derived from rat substantia nigra by inhibiting VMAT-2 with reserpine to increase free cytosolic dopamine concentration, to study aminochrome-dependent neurotoxicity in the absence of exogenous oxidizing agents such as metals, which may potentiate an aminochrome cytotoxic effect. The expression of VMAT-2 in RCSN-3 cells was determined by reverse transcriptase-polymerase chain reaction and immunocytochemistry. We observed double membrane bodies containing melanin when RCSN-3 cells were incubated with 100 microM dopamine by using transmission electron microscopy. No significant difference in the cell death was observed when the cells were treated 100 microM dopamine and 25 microM reserpine in the absence or presence of 100 microM dicoumarol, an inhibitor of DT-diaphorase. The lack of effect was due to the inhibitory action of 25 microM reserpine on DT-diaphorase (Ki = 24 microM). However, a significant increase in the cell death was observed when DT-diaphorase was inhibited when the cells were incubated with 1 microM reserpine and 100 microM dopamine for 12 h since at this concentration reserpine inhibits VMAT-2 but not DT-diaphorase. Under this condition, we observed (i) the formation of blebbing; (ii) chromatin condensation accompanied by the formation of massive patches in contact with the nuclear membrane; (iii) the smoothness of the cell's surface, that is, lack of surface microprojections; and (iv) mitochondrial damage characterized by disruption of cristae architecture, which remains closely packed; disorganization of the mitochondrial matrix due to separation of the outer membrane from the internal membrane and considerable enlargement of the intermembrane space; and disruption of the external mitochondrial membrane determined by transmission electron microscopy. These results support the proposed neuroprotective role of DT-diaphorase against aminochrome neurotoxicity, and it suggests that RCSN-3 cells incubated with reserpine and dopamine are an excellent and more physiological cellular experimental model to study the role of dopamine oxidation in neurotoxic effects of dopamine.

Isolation, transmission and purification of the High Plains virus

The wheat curl mite ( Aceria tosichella Keifer) often simultaneously transmits the High Plains virus and Wheat streak mosaic virus under field conditions, resulting in doubly infected plants. In this study, a pure culture of the High Plains virus (isolate HPV95ID), which was infected with both High Plains virus and Wheat streak mosaic virus, was mechanically transmitted from barley ( Hordeum vulgáre L.) to maize ( Zea mays L.) by vascular puncture inoculation. Different water temperatures and durations for soaking kernels at pre-inoculation and different incubation temperatures and durations at post-inoculation on transmission of High Plains virus were studied. Transmissions of the High Plains virus were significantly different for post-inoculation incubations at 11, 21, or 30 °C after a 2 h pre-inoculation soaking at 30 °C and post-inoculation incubations of kernels for 1 day versus 2 days. Use of Cs 2SO 4 in a partial purification protocol resulted in infectious final fractions. Bioassays, serological assays, analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and examinations by electron microscopy confirmed isolation of a pure culture of High Plains virus from infectious final partially purified fractions. We demonstrate infectivity of the final fractions and associate it with the High Plains disease symptoms, the 32 kDa protein and double membrane bodies and discuss this evidence to support the viral nature of High Plains virus.

Ultrastructure of a hypovirulent isolate of Sclerotinia sclerotiorum containing double-stranded RNA

Sclerotia and hyphae of hypovirulent double-stranded RNA (dsRNA)-containing and virulent dsRNA-free isolates of Sclerotinia sclerotiorum were examined to describe and compare their subcellular structures using light and electron microscopy. Light microscopic analysis of virulent and hypovirulent tissues indicated that, in general, tissues from both isolates were similar. However, sclerotia of the hypovirulent isolate contained fewer protein bodies than those of the virulent isolate and both hyphae and sclerotia of the hypovirulent isolate displayed a more granular appearance throughout the cytoplasm. Electron microscopic analysis revealed that the granular appearance was associated with the presence of many small, double membrane bodies within all cells of the hypovirulent isolate. Comparable structures were not observed in any tissues examined from the virulent isolate. The average diameter of the double membrane bodies was 70 nm and they often were observed in continuum with the nuclear envelope. Particles representative of a mycovirus were not observed in any virulent or hypovirulent tissues. The results indicate that double-stranded RNA and hypovirulence in S. sclerotiorum is not associated with a typical mycovirus but may represent an unencapsidated mycovirus or viral-like agent packaged as double membrane bodies.

Abstracts of Papers Presented at the Annual Meeting of the Canadian Phytopathological Society Banff, Alberta June 1991

Abstracts of Papers Presented at the Annual Meeting of the Canadian Phytopathological Society Banff, Alberta June 1991

Ultrastructure of Plant Leaf Tissue Infected with Mite-Borne Viral-Like Pathogens

A pathogen transmitted by the eriophyid mite, Aceria tulipae, infects a number of Gramineae producing symptoms similar to wheat spot mosaic virus (1). An electron microscope study of leaf ultrastructure from systemically infected Zea mays, Hordeum vulgare, and Triticum aestivum showed the presence of ovoid, double membrane bodies (0.1 - 0.2 microns) in the cytoplasm of parenchyma, phloem and epidermis cells (Fig. 1 ).