Tomato Spotted Wilt Virus RNA Research Articles

Rapid Extraction of Plant Nucleic Acids by Microneedle Patch for In-Field Detection of Plant Pathogens.

Plant diseases pose a significant threat to global food security. Molecular diagnosis currently plays a crucial role in mitigating the negative impacts of plant diseases by accurately identifying the disease-causing pathogens and revealing their genotypes. However, current molecular assays are constrained to the laboratory because of the cumbersome protocols involved in plant nucleic acid extraction. To streamline this, we have developed a polymeric microneedle (MN) patch-based nucleic acid extraction method, which can be applied to various plant tissues and easily performed in field settings without using bulky laboratory equipment. The MN patch instantly isolates both host and pathogen's DNA and RNA from plant leaves by two simple steps: press and rinse with a buffer solution or nuclease-free water. The MN-extracted DNA and RNA are purification-free and directly applicable to downstream molecular assays such as polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Here, we describe the fabrication procedures of the MN patch and demonstrate the application of the MN method by extracting Phytophthora infestans DNA and tomato spotted wilt virus (TSWV) RNA from infected tomato leaves. After MN extraction, we directly utilize the MN-extracted nucleic acid samples to run PCR, RT-PCR, LAMP, or RT-LAMP reactions to amplify various biomarker genes, such as the ribulose-bisphosphate carboxylase (rbcL) gene of host tomato DNA, internal transcribed spacer (ITS) region of P. infestans DNA, and nucleocapsid (N) gene of TSWV RNA. Furthermore, this simple and rapid nucleic acid method can be integrated with portable nucleic acid amplification platforms such as smartphone-based microscopy devices to achieve "sample-to-answer" detection of plant pathogens directly in the field.

A Tomato Spotted Wilt Virus S RNA-based Replicon System in Yeast

Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus of the order Bunyavirales, family Tospoviridae, genus Orthotospovirus. TSWV infects a broad range of plant species, causing serious economic losses. Despite its agronomic importance, molecular biological understanding of TSWV has been limited, partly due to the lack of a reverse genetics system, which would enable genetic manipulation of the virus. Here, we report that RNA synthesis by TSWV RNA polymerase occurs in the yeast Saccharomyces cerevisiae using a segment of the TSWV genome, S RNA expressed from cloned cDNA, as a template. Viral nucleocapsid protein was required for RNA synthesis. Replacement of the protein-coding and intergenic regions of TSWV S RNA by a yellow fluorescent protein (YFP)-coding sequence drastically increased the accumulation of both sense and antisense strands of the RNA, showing that this RNA was replicated. Using this system, we revealed that efficient RNA synthesis by TSWV RNA polymerase in yeast requires the 5′-terminal 17-nt and 3′-terminal ~50-nt regions of the TSWV S cRNA (complementary RNA to the genomic RNA) template.

Effect of Spinosad Resistance on Transmission of Tomato Spotted Wilt Virus by the Western Flower Thrips (Thysanoptera: Thripidae).

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis (Pergande) in a persistent-propagative manner. We previously observed significant results in terms of feeding behavior of spinosad-susceptible (Ivf03) and -resistant (Spin-R) strains of F. occidentalis using electrical penetration graph. TSWV transmission by the two strains was compared in the present study. The results showed that the titer of TSWV-N RNA (a part of S RNA of TSWV and encoding the nucleocapsid protein) in Ivf03 and Spin-R strains was not significantly different after a 48-h inoculation access period. The TSWV transmission rate did not significantly differ between the two strains and was 51.0% for Ivf03 and 44.4% for Spin-R. The virus transmission rate was significantly higher for males than females of both strains. The virus transmission rate for males and females of Ivf03 was 68.1 and 33.8%, respectively; however, in case of Spin-R, it was 60 and 28.8% for males and females, respectively. Additionally, number of probes and duration of probes were generally greater for viruliferous females of Ivf03 than for viruliferous females of Spin-R but the total number and duration of noningestion probes did not significantly differ between males of the two strains. The latter finding behavior may help explain the similar transmission rates for the susceptible and resistant strains.

Diversity of Thrips Species and Vectors of Tomato Spotted Wilt Virus in Tomato Production Systems in Kenya.

Thrips have been recognized as primary vectors of tomato spotted wilt virus (TSWV) with Frankliniella occidentalis (Pergande) reported as the most important and efficient vector, while other species such as Thrips tabaci Lindeman also include populations that can vector the virus. A study was undertaken to establish the diversity of thrips and presence of vectors for TSWV in four major tomato production areas in Kenya. The cytochrome oxidase 1 (CO1) gene was used to generate sequences from thrips samples collected from tomatoes and weeds, and phylogenetic analysis done to establish the variation within potential vector populations. Ceratothripoides brunneus Bagnall was the predominant species of thrips in all areas. F. occidentalis and T. tabaci were abundant in Nakuru, Kirinyaga, and Loitokitok but not detected at Bungoma. Other vectors of tospoviruses identified in low numbers were Frankliniella schultzei (Trybom) and Scirtothrips dorsalis Hood. Variation was observed in T. tabaci, F. occidentalis, and F. schultzei. Kenyan specimens of T. tabaci from tomato belonged to the arrhenotokous group, while those of F. occidentalis clustered with the Western flower thrips G group. The detection of RNA of TSWV in both of these species of thrips supported the role they play as vectors. The study has demonstrated the high diversity of thrips species in tomato production and the occurrence of important vectors of TSWV and other tospoviruses.

Possible involvement of eEF1A in Tomato spotted wilt virus RNA synthesis

Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus in the family Bunyaviridae and propagates in both insects and plants. Although TSWV can infect a wide range of plant species, host factors involved in viral RNA synthesis of TSWV in plants have not been characterized. In this report, we demonstrate that the cell-free extract derived from one of the host plants can activate mRNA transcriptional activity of TSWV. Based on activity-guided fractionation of the cell-free extract, we identified eukaryotic elongation factor (eEF) 1A as a possible host factor facilitating TSWV transcription and replication. The RNA synthesis-supporting activity decreased in the presence of an eEF1A inhibitor, suggesting that eEF1A plays an important role in RNA synthesis of TSWV.

Phylogenetic and recombination analysis of tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) severely damages and reduces the yield of many economically important plants worldwide. In this study, we determined the whole-genome sequences of 10 TSWV isolates recently identified from various regions and hosts in Korea. Phylogenetic analysis of these 10 isolates as well as the three previously sequenced isolates indicated that the 13 Korean TSWV isolates could be divided into two groups reflecting either two different origins or divergences of Korean TSWV isolates. In addition, the complete nucleotide sequences for the 13 Korean TSWV isolates along with previously sequenced TSWV RNA segments from Korea and other countries were subjected to phylogenetic and recombination analysis. The phylogenetic analysis indicated that both the RNA L and RNA M segments of most Korean isolates might have originated in Western Europe and North America but that the RNA S segments for all Korean isolates might have originated in China and Japan. Recombination analysis identified a total of 12 recombination events among all isolates and segments and five recombination events among the 13 Korea isolates; among the five recombinants from Korea, three contained the whole RNA L segment, suggesting reassortment rather than recombination. Our analyses provide evidence that both recombination and reassortment have contributed to the molecular diversity of TSWV.

The effect of virus titre on acquisition efficiency of Tomato spotted wilt virus by Frankliniella occidentalis and the effect of temperature on detectable period of the virus in dead bodies

The acquisition efficiency of Tomato spotted wilt virus (TSWV) by Frankliniella occidentalis was examined using TSWV-infected Datura stramonium with various virus titres. TSWV quantities in leaves were measured using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The percentages of viruliferous F. occidentalis significantly correlated with DAS-ELISA and qRT-PCR values in the fed leaf piece. The detectable period of TSWV in viruliferous F. occidentalis adults trapped on sticky traps was also examined at various temperatures. At 25°C or less, TSWV could still be detected by DAS-ELISA in the bodies for at least 20 days after the capture of viruliferous F. occidentalis, although ELISA values had decreased gradually over time more rapidly at higher temperatures. The quantities of TSWV RNA detected by qRT-PCR rapidly decreased. The mean value decreased to half in a day, and reduced to 7.3% of the initial mean value after 14 days.

Viral genetic determinants for thrips transmission of Tomato spotted wilt virus

Tomato spotted wilt virus (TSWV) is transmitted exclusively by thrips in nature. A reassortment-based viral genetic system was used to map transmissibility by thrips to the medium (M) RNA of TSWV. To locate determinants of thrips transmission in the M RNA, 30 single-lesion isolates (SLIs) were generated from a single TSWV isolate that was inefficiently transmitted by thrips. Three of the 30 SLIs were transmitted by thrips, and 27 were not. Sequence analysis of the M RNA, thrips transmissibility assays, G(C) protein analysis, and transmission electron microscopic studies revealed that a specific nonsynonymous mutation (C1375A) in the G(N)/G(C) ORF of the M RNA resulted in the loss of thrips transmissibility without inhibition of virion assembly. This was in contrast to other nontransmissible SLIs, which had frameshift and/or nonsense mutations in the G(N)/G(C) ORF but were defective in virion assembly. The G(C) glycoprotein was detectable in the C1375A mutants but not in the frameshift or nonsense mutants. We report a specific viral determinant associated with virus transmission by thrips. In addition, the loss of transmissibility was associated with the accumulation of defective haplotypes in the population, which are not transmissible by thrips, rather than with the presence of a dominant haplotype that is inefficiently transmitted by thrips. These results also indicate that the glycoproteins may not be required for TSWV infection of plant hosts but are required for transmissibility by thrips.

Winter Occurance and Spring Migration of Frankliniella fusca (Hinds) (Thysanoptera: Tripidae) in North Carolina Peanut (Arachis hypogaea L.) Fields

Abstract Overwintering of Frankliniella fusca (Hinds), tobacco thrips, in North Carolina and their subsequent spring movement into peanut fields were evaluated using two winter sampling techniques and three spring sampling techniques at the Peanut Belt Res. Sta., Lewiston, NC. In the spring of 1992 and 1993, for 14 d following peanut planting, the aerial movement of tobacco thrips was monitored using cylindrical sticky traps, trap plants, and exclusion cages. Frankliniellafusca were trapped significantly more often at 0.2 m and 0.9 m than at 1.8 m and during the afternoons. Thrips catch was significantly influenced by prevailing wind direction. No thrips were caught on sticky traps on days when maximum temperatures did not exceed 18.7 C. Frankliniella fusca began to colonize emerging peanut plants as they cracked the soil surface on days when there were temperatures above 18.7 C and times when there was no precipitation. Aerial F. fusca movement was monitored with sticky traps for three consecutive winters during 1993–96. Tobacco thrips were collected on sticky traps throughout the winter; however, counts were lower in months and years with lower temperatures. Tobacco thrips, caged throughout the winter with peanut plants infected with tomato spotted wilt virus (TSWV), were analyzed for the presence of a nonstructural protein (NSs) encoded for by the small RNA of TSWV and infectivity by ELISA. A total of eight tobacco thrips were collected, of which one tested positive.

Dynamics of Tomato spotted wilt virus Replication in the Alimentary Canal of Two Thrips Species

ABSTRACT Transmission of Tomato spotted wilt virus (TSWV) is dependent on virus uptake in the midgut prior to virus movement to the salivary glands. Replication of TSWV in the alimentary canal of tobacco thrips (TT, Frankliniella fusca) and western flower thrips (WFT, F. occidentalis) was investigated by immunolocalization of the nonstructural protein (NSs) encoded by the small RNA of TSWV and fluorescence microscopy. Analysis of cohorts during development from larva to adults following virus acquisition by first instar larva indicated that virus replication followed a specific time-course pattern in the foregut, regions of the midgut, salivary glands, and ligaments between the midgut and salivary glands. Initial virus replication occurred only in epithelial cells of midgut-1 but, upon infection of muscle cells, the virus moved to the midgut-2, foregut, midgut-3, and salivary glands. The ligaments between the midgut and salivary glands appeared to be a route for virus to invade the salivary glands. No virus replication was observed in the hindgut, Malpighian tubules, or tubular salivary glands. The dynamics of TSWV replication, as measured by NSs accumulation, were similar in both TT and WFT.

In vivo analysis of the TSWV cap-snatching mechanism: single base complementarity and primer length requirements.

Requirements for capped leader sequences for use during transcription initiation by tomato spotted wilt virus (TSWV) were tested using mutant alfalfa mosaic virus (AMV) RNAs as specific cap donors in transgenic Nicotiana tabacum plants expressing the AMV replicase proteins. Using a series of AMV RNA3 mutants modified in either the 5'-non-translated region or in the subgenomic RNA4 leader, sequence analysis revealed that cleaved leader lengths could vary between 13 and 18 nucleotides. Cleavage occurred preferentially at an A residue, suggesting a requirement for a single base complementarity with the TSWV RNA template, which could be confirmed by analyses of host mRNAs used in vivo as cap donors.

An RNA-Dependent RNA Polymerase Activity Associated with Virions of Tomato Spotted Wilt Virus, a Plant- and Insect-Infecting Bunyavirus

An RNA-dependent RNA polymerase activity was found associated with virions of tomato spotted wilt virus (TSWV), a plant- and insect-infecting member of the family Bunyaviridae. Radiolabeled nucleoside triphosphates were incorporated into trichloroacetic acid-precipitable products by detergent-disrupted, purified TSWV virions. Incorporation was reduced to near-background levels when RNase was present in the reaction mixture. The predominantly double-stranded RNA products were RNase-resistant at high but not low salt concentrations. The activity required manganese and was independent of a DNA template. Discrete products of approximately 3.0 kb and heterogeneous smaller products were synthesized that hybridized to purified TSWV RNA and transcripts of cDNA clones encompassing parts of each of the three genomic RNAs. The predominant products were viral sense although significant amounts of viral complementary sense S RNA products were also synthesized.

Dot Blot Detection of Tomato Spotted Wilt Virus RNA in Plant and Thrips Tissues by cDNA Clones

Two plasmids, pTSWV7 and pTSWV80, containing inserts of 460 and 870 bp, respectively, were characterized for specificity to TSWV RNA and were selected to develop a diagnostic dot blot assay. 32P-labeled hybridization probes from these clones can detect TSWV in as little as 16 ng of total RNA from tobacco, 80 ng from tomato and lettuce, and 400 ng from chrysanthemum and pepper

Evidence that Tomato Spotted Wilt Virus RNA is a Positive Strand

SUMMARY RNA of tomato spotted wilt virus (TSWV) was analysed by gel electrophoresis on methylmercury-agarose gels. Three segments were detected with mol. wt. of 2.7 × 106, 1.7 × 106 and 1.1 × 106. The behaviour on oligo(dT)-cellulose columns indicated that TSWV RNA does not contain poly(A) sequences. TSWV RNA directed the synthesis of several proteins in a wheatgerm cell-free system and a messenger-dependent rabbit reticulocyte lysate. Analysis of the translation products by gel electrophoresis revealed that two major polypeptides were synthesized in both systems. One could be identified by immunoprecipitation as the nucleocapsid protein; the identity of the other, having a mol. wt. of 60000, is unknown. The above results demonstrate that TSWV contains a positive-stranded RNA and this conclusion was confirmed by other experiments. TSWV RNA labelled with 125I did not hybridize with polyribosomal RNA from infected leaves and transcriptase activity could not be detected in purified preparations of TSWV or in nucleocapsid extracts from infected leaves. This study supports the view that TSWV is a unique membrane-bounded plant virus whose genome consists of three RNA segments which act as mRNA.