Ultrastructural effects of infection caused by Tobacco rattle virus transmitted by Trichodorus primitivus in potato and tobacco tissues

The studies focus on an ultrastructural analysis of the phenomenon of intercellular and systemic (vascular) transport of tobacco rattle virus (TRV) in tissues of the infected plants. TRV is a dangerous pathogen of cultivated and ornamental plants due to its wide range of plant hosts and continuous transmission by vectors—ectoparasitic nematodes. Two weeks after infection with the PSG strain of TRV, tobacco plants of the Samsun variety and potato plants of the Glada variety responded with spot surface necroses on inoculated leaf blades. Four weeks after the infection a typical systemic response was observed on tobacco and potato leaves, necroses on stems and lesions referred to as corky ringspot. Ultrastructural analysis revealed the presence of two types of TRV virions: capsidated and non-capsidated forms in tobacco and potato tissues. In the protoplast area, viral particles either occurred in a dispersed form or they formed organised inclusions of virions. We demonstrated for the first time the presence of non-capsidated-type TRV in the vicinity of and inside plasmodesmata. Capsidated particles of TRV were observed in intercellular spaces of the tissues of aboveground and underground organs. Expanded apoplast area was noted at the cell wall, with numerous dispersed non-capsidated-type TRV particles. These phenomena suggest active intercellular transport. Our ultrastructure studies showed for the first time that xylem can be a possible route of TRV systemic transport. We demonstrated that both capsidated and non-capsidated virions, of varied length, participate in long-distance transport. TRV virions were more often documented in xylem (tracheary elements and parenchyma) than in phloem. Non-capsidated TRV particles were observed inside tracheary elements in a dispersed form and in regular arrangements in potato and tobacco xylem. The presence of TRV virions inside the bordered pits was demonstrated in aboveground organs and in the root of the tested plants. We documented that both forms of TRV virions can be transported systemically via tracheary elements of xylem.

The evidence of Tobacco rattle virus impact on host plant organelles ultrastructure

The one‐third of plant viruses are seed transmitted, and this has significant economic consequences. Tobacco rattle virus (TRV), belonging to the genus Tobravirus and family Virgaviridae, has one of the widest host range of any known plant viruses. TRV infects vegetative organ and effects seed and pollen development that results in a decrease in crop yield. The mechanisms by which Tobravirus is transmissible to seeds are still poorly understood. The presence of the virus in pollen grains and inside ovaries is linked with seed transmission and can have effects on virus particles' transport during the pollination and fertilization process. This paper focuses on the significant impact of TRV on pepper and tobacco anthers and ultrastructure changes in ovaries. The presence of two types of TRV particles in ovary wall parenchyma and vascular tissues as well as in placenta cells was demonstrated via ultrastructural analysis. For the first time, the regular inclusion of virus particles was reported in both ovule integuments and nucellus parenchyma cells. Immunolocalization of TRV capsid proteins indicated the deposition of TRV CP epitope in ovary vascular bundles and in placenta cells. Moreover, the presence of virus particles was demonstrated inside pepper seeds in endothelium and integument parenchyma layers as well as on the embryo cell wall. Virus particles were found not only on the surface of pollen grains but also inside pepper pollen protoplasts in mature anthers. Also, this is the first time where TRV particles are reported in both differentiated endothecium cells and the remaining tapetum cells. Moreover, the detection of TRV capsid protein epitope in tobacco and pepper vascular anther tissues as well as in tapetum and endothecium cells was correlated with TRV distribution in infected anthers. Demonstrated analyses indicated that pollen grains and ovaries with ovules as well as could be a natural source of TRV transmission.

Serotypes and particle dimensions of tobacco rattle viruses from Europe and America

Weigela (Weigela florida (Bunge) A. DC., Family: Caprifoliaceae) are woody shrubs native to North China, Korea, and Japan. In the U.S., weigela are commonly used as landscape ornamental plants (McNamara et al. 2010). Two viruses have been reported in weigela: tomato spotted wilt orthotospovirus and impatiens necrotic spot orthotospovirus (Sastry et al. 2019). Ten weigela plants, originating from commercial nurseries in Minnesota, exhibiting chlorosis, chlorotic line patterns, and necrosis (e-Xtra) were submitted for virus diagnostics as potted plants at the University of Minnesota Plant Disease Clinic and the Virology Lab in 2019 and 2020 (five plants each year). Under greenhouse conditions, symptoms progressed from chlorosis to necrosis and even plant death in two of the five plants in 2019. Electron microscopy revealed rod-shaped particles of ≈20 nm in diameter and lengths between 40-200 nm with similar morphology to members of the genus Tobravirus (e-Xtra). Virus-like particles were enriched by ultracentrifugation and total nucleic acids were extracted from partial purifications using a phenol:chloroform extraction method (Lockhart et al. 1997). Tobacco rattle virus (TRV) was identified by cloning and sequencing of the 463bp amplicon obtained with the TRV detection primers described in Robinson, 1992. High-throughput sequencing (HTS) was done to confirm the TRV detection. A cDNA library was prepared from purified viral RNA using the TruSeq Stranded mRNA kit and sequenced on Illumina NovaSeq 6000 platform as 150 bp-paired end reads. A total of 44,316,446 raw data reads were obtained, preprocessed using the BBDuk plugin, and de novo assembled using SPAdes assembler. Viral contigs were identified using the NCBI BLASTX tool. The assembly of TRV RNA1 was 6,842 nt with 20,627,348 reads (47% of total reads) mapped to it and an average coverage per nucleotide at 323,639X. The assembly of TRV RNA2 was 3,033 nt with 22,769,253 reads (52% of total reads) mapped to it and an average coverage per nucleotide at 798,660X. NCBI GenBank accession numbers for the assemblies representing RNA1 and RNA2 are OQ408335 and OQ408336, respectively. NCBI BLASTn analysis showed the highest level of nucleotide identity to TRV genomic RNA segments 1 and 2, with 97% and 99% identity to the TRV isolate RNA1 (GQ903771) and RNA2 (GQ903772), respectively, that originated from Michigan potato. No other viral contigs were detected from the virion nucleic acid extraction by HTS, however this enrichment method doesn't exclude other viruses. In addition to using the detection primers by Robinson 1992, we designed primers based on our HTS data: TRV-WG-DetF3 5'- GACGAAGGAGGCTGTCATTGC-3' and TRV-WG-DetR3 5'-CGGACTATCGTGATGCCCATGC- 3'. RT-PCR amplicons from each of the 10 symptomatic plants were cloned and sequenced. Among these clones, Sanger sequence identities ranged between 96-100% compared to the HTS data and 98-99% to the TRV potato isolate from MI. To our knowledge, this is the first report of TRV infecting the ornamental host W. florida worldwide. TRV is a nematode-transmitted viral pathogen of economic importance, most notably in potatoes (Sastry et al. 2019). In the US, TRV has been reported on several landscape ornamentals, horticultural crops, and native habitats. Further research is needed to investigate the impact of TRV on the ornamental industry and the role of ornamentals as reservoirs for cultivated crops like potatoes.

SUMMARY Electron microscopy of ultrathin sections indicated that nucleoprotein particles of raspberry ringspot (RRV) and tobacco rattle (strain CAM; TRV-CAM) viruses occurred in mixed aggregates in cells of doubly infected Nicotiana benthamiana leaves, and in protoplasts doubly infected by inoculation. RRV particles were attached to the sides and ends of TRV-CAM particles that were mostly bound to mitochondria. RRV particles attached to TRV-CAM particles linking mitochondria in clusters are considered to be the structures represented by the aggregates of RRV particle antigen found previously by fluorescent antibody staining in doubly infected protoplasts but not in those infected with RRV alone.

SUMMARY The addition of actinomycin D (25 µg/ml) or cordycepin (1 mm) to protoplast cultures immediately after inoculation with particles of tobacco mosaic, tobacco rattle, tobacco ringspot or potato leafroll viruses resulted in a decrease in the proportion of protoplasts becoming infected, as judged by staining with fluorescent antibody to virus particles. A delay of a few hours between the inoculation and the addition of either inhibitor largely or completely eliminated this effect. In contrast, infection was unaffected by the addition of actinomycin D when the protoplasts were inoculated with RNA preparations from tobacco mosaic or tobacco rattle viruses.

Sixteen isolates of Tobacco rattle virus (TRV) obtained from the United States and Canada were characterized in the present study. Nucleotide-sequence analysis of open reading frame 4 (ORF4) in RNA1 of seven test isolates revealed a high degree of genetic diversity. Potato isolates of TRV clustered in several distinct groups, with isolates from the same geographic region falling into different groups. Phylogenetic analysis of TRV isolates based on nucleotide sequences and on restriction fragment length polymorphism (RFLP) produced similar groupings. TRV was detected in various potato tissues of inoculated, field-grown plants, including progeny tubers, leaves, and roots, by reverse transcription – polymerase chain reaction (RT–PCR), using a pair of TRV-specific primers targeting ORF4. TRV RNA was detected in highly diluted total RNA preparations (1 / 15 625) and in composite tuber samples (200–400 tubers). RT–PCR followed by RFLP analysis was shown to be an efficient procedure for specific and sensitive detection and identification of TRV in potato.

The use of negative staining in the electron microscopic examination of plant viruses in crude extracts

Transgenic Samsun NN tobacco plants expressing the coat protein of tobacco rattle virus were exposed to mechanical leaf inoculation with tobacco rattle virus and to viruliferous trichodorid vector nematodes. Whereas plants were resistant to mechanical inoculation the vector nematodes successfully transmitted tobacco rattle virus to the roots as well as to the leaves of these plants. It is suggested that transgenic resistance is overcome either because vector nematodes inject relatively large numbers of virus particles into a cell or because they inject destabilized particles. The results indicate that coat protein-mediated resistance is unlikely to be of value for controlling tobacco rattle virus in field crops.

Plant virus uncoating as a result of virus-cell wall interactions

SUMMARY 16 and N5 are naturally occurring tobravirus isolates that produce symptoms in herbaceous plants similar to those induced by strains of tobacco rattle virus (TRV). In immunosorbent electron microscopy tests, however, they reacted with antisera to particles of pea early-browning virus (PEBV), not TRV. Furthermore, these tests indicated that 16 was related to the British serotype of PEBV and N5 to the Dutch. Pseudo-recombinant isolates were produced by reassortment of the genome parts of 16 or N5 with those of TRV, in any combination, but not in most combinations with those of PEBV. However, 16 RNA-2 was replicated in plants inoculated also with RNA-1 from an isolate of the British serotype of PEBV, but the PEBV RNA-1 was imperfectly packaged by 16 coat protein, and the virus particles seemed to have only limited stability. Nucleic acid hybridization experiments showed that the RNA-1 sequences of both 16 and N5 were similar to those of TRV strains. 16 RNA-2 contained sequences resembling those of the British serotype of PEBV, but with some TRV-like sequences at the 3′ and 5′ ends, whereas N5 RNA-2 contained more extensive TRV-like 3′ and 5′ ends flanking sequences that were related, but perhaps not closely, to those of the Dutch serotype of PEBV. Thus, the RNA-2 species of 16 and N5 were recombinant molecules that contained sequences typical of both TRV and PEBV, and which probably had separate but similar evolutionary origins. As a result of their hybrid nature, 16 and N5 were part of the gene pool and had the pathogenicity of TRV, while possessing the serological properties of PEBV.

Yellow ringspotting and concentric line patterns in plants of Dicentra (bleeding heart), Epimedium (barrenwort), and Heuchera (coral bells) from commercial nurseries and home gardens in Minnesota, Michigan, and Massachusetts were associated with infection by Tobacco rattle virus (TRV), which was identified by particle morphology, enzyme-linked immunosorbent assay and immunosorbent electron microscopy. No other viruslike particles were observed by electron microscopy in partially purified preparations of TRV-infected leaf tissue, and TRV was not detected in asymptomatic plants. This is the first report of TRV occurrence in Dicentra in the United States and the first report of TRV occurrence in Epimedium and Heuchera. In previous reports (1,2) we have called attention to the increasing incidence of TRV in vegetatively propagated perennial ornamental plant species in the United States and to the potential for virus spread to crops such as potato, in which TRV has not been reported in the midwestern United States. It is possible that increased international trade in vegetatively propagated ornamental plants may be resulting in the introduction of TRV and other exotic viruses into the United States and elsewhere. It is also possible that the natural occurrence of TRV in North America may be actually more widespread than has been reported.

SUMMARY Non-crystalline inclusions, referred to as X-bodies, developed in leaf-hair cells of Nicotiana clevelandii infected with a RNA-producing defective isolate (cam/df) of tobacco rattle virus but not in those infected with the nucleo- protein particle-producing strain (cam) from which cam/df was derived. The X-bodies appeared 3 days after inoculation; some persisted for at least 10 weeks. They were digested by pronase and were rich in RNA, which was protected by proteinaceous material from digestion by pancreatic ribo- nuclease; they did not contain virus coat-protein detectable by fluorescent antibody tests. Electron microscopy of thin sections showed that mitochondria in infected cells developed two abnormalities—peripheral membranous sacs and membrane bounded vacuoles—and then aggregated, together with ribosomes and material containing small darkly staining granules, to form small X- bodies. These fused to form larger X-bodies, in which the mitochondria were further modified and eventually became barely recognizable. Finally, the contents of the X-bodies became increasingly amorphous, and the bodies disintegrated. X-bodies also developed in leaf-hair cells infected with the virus particle- producing strain prn, but were less common and mostly persisted for only a few days. These X-bodies were formed in the same way as those produced by cam/df, but contained in addition small aggregates of virus particles resembling the aggregates found in the cytoplasm in other parts of the same cells. The frequency of formation, and persistence, of X-bodies induced by these tobacco rattle viruses paralleled the severity and persistence of their macroscopic effects on N. clevelandii. The possibility that mitochondria are sites of synthesis of tobacco rattle virus RNA is discussed.

The occurrence of Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV) Tobacco rattle virus (TRV) in gladiolus, iris, tulip and Iris yellow spot virus (IYSV) in iris was investigated by examining the plants by the means of serological techniques (ELISA). ELISA was applied to determine the presence of BYMV, CMV, TRV infections in both aerial and underground parts of gladiolus, iris, and tulip, and IYSV on the aerial parts of iris, respectively. 262 gladiolus plants were tested. 63.7% was infected by BYMV, 29.4 % by CMV, and 2.7 % by TRV. Out of 180 plants of iris, 1.1% was infected by BYMV, 6.7% by CMV, 2.8% by TRV, and 0% by IYSV. Out of 28 plants of tulip, 28.6% was infected by CMV, and 7.1% by TRV. ELISA proved to be a suitable method for detection of viruses in leaves of these ornamental plants, but it often failed to detect viruses in flowers and corms. A high transmission of BYMV by gladiolus cormlets was also found.