Next-generation sequencing identification and multiplex RT-PCR detection for viruses infecting cigar and flue-cured tobacco.

BACKGROUND: Early, precise and simultaneous identification of plant viruses is of great significance for preventing virus spread and reducing losses in agricultural yields. METHODS AND RESULTS: In this study, the identification of plant viruses from symptomatic samples collected from a cigar tobacco planting area in Deyang and a flue-cured tobacco planting area in Luzhou city, Sichuan Province, China, was conducted by deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform, and plant virus-specific contigs were generated based on virus-derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis were performed to determine the species or strains of these viruses. A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples, with a total of 105 contigs mapped to the closest plant viruses with lengths ranging from 34 ~ 1720 nt. The results indicated that the major viruses were potato virus Y, Chilli veinal mottle virus, tobacco vein banding mosaic virus, tobacco mosaic virus and cucumber mosaic virus. Subsequently, a fast and sensitive multiplex reverse transcription polymerase chain reaction assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan. CONCLUSIONS: These results provide a theoretical basis and convenient methods for the rapid detection and control of viruses in cigar- and flue-cured tobacco.

Detection of plant virus particles with a capacitive field-effect sensor.

Plant viruses are major contributors to crop losses and induce high economic costs worldwide. For reliable, on-site and early detection of plant viral diseases, portable biosensors are of great interest. In this study, a field-effect SiO2-gate electrolyte-insulator-semiconductor (EIS) sensor was utilized for the label-free electrostatic detection of tobacco mosaic virus (TMV) particles as a model plant pathogen. The capacitive EIS sensor has been characterized regarding its TMV sensitivity by means of constant-capacitance method. The EIS sensor was able to detect biotinylated TMV particles from a solution with a TMV concentration as low as 0.025 nM. A good correlation between the registered EIS sensor signal and the density of adsorbed TMV particles assessed from scanning electron microscopy images of the SiO2-gate chip surface was observed. Additionally, the isoelectric point of the biotinylated TMV particles was determined via zeta potential measurements and the influence of ionic strength of the measurement solution on the TMV-modified EIS sensor signal has been studied.

Dual targeting of a virus movement protein to ER and plasma membrane subdomains is essential for plasmodesmata localization.

Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MPFMV) using live cell imaging, focusing on its ER-directing signal peptide (SPFMV). Transiently expressed MPFMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SPFMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MPFMV mutant lacking SPFMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MPFMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with Arabidopsis synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SPFMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MPFMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MPFMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MPFMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MPFMV into PD.

Spicing Up the N Gene: F. O. Holmes and Tobacco mosaic virus Resistance in Capsicum and Nicotiana Plants.

One of the seminal events in plant pathology was the discovery by Francis O. Holmes that necrotic local lesions induced on certain species of Nicotiana following rub-inoculation of Tobacco mosaic virus (TMV) was due to a specific interaction involving a dominant host gene (N). From this, Holmes had an idea that if the N gene from N. glutinosa was introgressed into susceptible tobacco, the greatly reduced titer of TMV would, by extension, prevent subsequent infection of tomato and pepper plants by field workers whose hands were contaminated with TMV from their use of chewing and smoking tobacco. The ultimate outcome has many surprising twists and turns, including Holmes' failure to obtain fertile crosses of N. glutinosa × N. tabacum after 3 years of intensive work. Progress was made with N. digluta, a rare amphidiploid that was readily crossed with N. tabacum. And, importantly, the first demonstration by Holmes of the utility of interspecies hybridization for virus resistance was made with Capsicum (pepper) species with the identification of the L gene in Tabasco pepper, that he introgressed into commercial bell pepper varieties. Holmes' findings are important as they predate Flor's gene-for-gene hypothesis, show the use of interspecies hybridization for control of plant pathogens, and the use of the local lesion as a bioassay to monitor resistance events in crop plants.

Translocation of Rigid Rod-Shaped Virus through Various Solid-State Nanopores.

Nanopores have been used as a high throughput tool for characterizing individual biomolecules and nanoparticles. Here, we present the translocation of rigid rod-shaped tobacco mosaic virus (TMV) through solid-state nanopores. Interestingly, due to the high rigidity of TMV, three types of events with distinctive characteristics at the capture process and a strong current fluctuation during the translocation of TMV are observed. A kinetic model is then proposed to address the dynamics of the translocation, followed by corresponding dynamics simulations. The results reveal that TMV has to rotate to fit and pass the pore when it is captured by a nanopore with an angle larger than the maximum angle that allows it to pass through. Then, we investigate the dependence of the rotation of TMV on the conductance fluctuations at the blockade stage. The results show that the rotation of TMV during the passage through the pore affects the current signal significantly. This study gives a fundamental understanding of the dynamics of rod-shaped particles translocating through the nanopore and how the current responds to it. It opens a new possible way to characterize the rigidity of analytes by nanopores.

Simple and portable magnetic immunoassay for rapid detection and sensitive quantification of plant viruses.

Plant pathogens cause major economic losses in the agricultural industry because late detection delays the implementation of measures that can prevent their dissemination. Sensitive and robust procedures for the rapid detection of plant pathogens are therefore required to reduce yield losses and the use of expensive, environmentally damaging chemicals. Here we describe a simple and portable system for the rapid detection of viral pathogens in infected plants based on immunofiltration, subsequent magnetic detection, and the quantification of magnetically labeled virus particles. Grapevine fanleaf virus (GFLV) was chosen as a model pathogen. Monoclonal antibodies recognizing the GFLV capsid protein were immobilized onto immunofiltration columns, and the same antibodies were linked to magnetic nanoparticles. GFLV was quantified by immunofiltration with magnetic labeling in a double-antibody sandwich configuration. A magnetic frequency mixing technique, in which a two-frequency magnetic excitation field was used to induce a sum frequency signal in the resonant detection coil, corresponding to the virus concentration within the immunofiltration column, was used for high-sensitivity quantification. We were able to measure GFLV concentrations in the range of 6 ng/ml to 20 µg/ml in less than 30 min. The magnetic immunoassay could also be adapted to detect other plant viruses, including Potato virus X and Tobacco mosaic virus, with detection limits of 2 to 60 ng/ml.

Towards instantaneous cellular level bio diagnosis: laser extraction and imaging of biological entities with conserved integrity and activity.

The prospect for spatial imaging with mass spectroscopy at the level of the cell requires new means of cell extraction to conserve molecular structure. To this aim, we demonstrate a new laser extraction process capable of extracting intact biological entities with conserved biological function. The method is based on the recently developed picosecond infrared laser (PIRL), designed specifically to provide matrix-free extraction by selectively exciting the water vibrational modes under the condition of ultrafast desorption by impulsive vibrational excitation (DIVE). The basic concept is to extract the constituent protein structures on the fastest impulsive limit for ablation to avoid excessive thermal heating of the proteins and to use strongly resonant 1-photon conditions to avoid multiphoton ionization and degradation of the sample integrity. With various microscope imaging and biochemical analysis methods, nanoscale single protein molecules, viruses, and cells in the ablation plume are found to be morphologically and functionally identical with their corresponding controls. This method provides a new means to resolve chemical activity within cells and is amenable to subcellular imaging with near-field approaches. The most important finding is the conserved nature of the extracted biological material within the laser ablation plume, which is fully consistent with in vivo structures and characteristics.

Single virus detection by means of atomic force microscopy in combination with advanced image analysis.

In the present contribution virions of five different virus species, namely Varicella-zoster virus, Porcine teschovirus, Tobacco mosaic virus, Coliphage M13 and Enterobacteria phage PsP3, are investigated using atomic force microscopy (AFM). From the resulting height images quantitative features like maximal height, area and volume of the viruses could be extracted and compared to reference values. Subsequently, these features were accompanied by image moments, which quantify the morphology of the virions. Both types of features could be utilized for an automatic discrimination of the five virus species. The accuracy of this classification model was 96.8%. Thus, a virus detection on a single-particle level using AFM images is possible. Due to the application of advanced image analysis the morphology could be quantified and used for further analysis. Here, an automatic recognition by means of a classification model could be achieved in a reliable and objective manner.

Application perspectives of localization microscopy in virology.

Localization microscopy approaches allowing an optical resolution down to the single-molecule level in fluorescence-labeled biostructures have already found a variety of applications in cell biology, as well as in virology. Here, we focus on some perspectives of a special localization microscopy embodiment, spectral precision distance/position determination microscopy (SPDM). SPDM permits the use of conventional fluorophores or fluorescent proteins together with standard sample preparation conditions employing an aqueous buffered milieu and typically monochromatic excitation. This allowed superresolution imaging and studies on the aggregation state of modified tobacco mosaic virus particles on the nanoscale with a single-molecule localization accuracy of better than 8 nm, using standard fluorescent dyes in the visible spectrum. To gain a better understanding of cell entry mechanisms during influenza A virus infection, SPDM was used in conjunction with algorithms for distance and cluster analyses to study changes in the distribution of virus particles themselves or in the distribution of infection-related proteins, the hepatocyte growth factor receptors, in the cell membrane on the single-molecule level. Not requiring TIRF (total internal reflection) illumination, SPDM was also applied to study the molecular arrangement of gp36.5/m164 glycoprotein (essentially associated with murine cytomegalovirus infection) in the endoplasmic reticulum and the nuclear membrane inside cells with single-molecule resolution. On the basis of the experimental evidence so far obtained, we finally discuss additional application perspectives of localization microscopy approaches for the fast detection and identification of viruses by multi-color SPDM and combinatorial oligonucleotide fluorescence in situ hybridization, as well as SPDM techniques for optimization of virus-based nanotools and biodetection devices.

Production of a de-novo designed antimicrobial peptide in Nicotiana benthamiana.

Antimicrobial peptides are important defense compounds of higher organisms that can be used as therapeutic agents against bacterial and/or viral infections. We designed several antimicrobial peptides containing hydrophobic and positively charged clusters that are active against plant and human pathogens. Especially peptide SP1-1 is highly active with a MIC value of 0.1 µg/ml against Xanthomonas vesicatoria, Pseudomonas corrugata and Pseudomonas syringae pv syringae. However, for commercial applications high amounts of peptide are necessary. The synthetic production of peptides is still quite expensive and, depending on the physico-chemical features, difficult. Therefore we developed a plant/tobacco mosaic virus-based production system following the 'full virus vector strategy' with the viral coat protein as fusion partner for the designed antimicrobial peptide. Infection of Nicotiana benthamiana plants with such recombinant virus resulted in production of huge amounts of virus particles presenting the peptides all over their surface. After extraction of recombinant virions, peptides were released from the coat protein by chemical cleavage. A protocol for purification of the antimicrobial peptides using high resolution chromatographic methods has been established. Finally, we yielded up to 0.025 mg of peptide per g of infected leaf biomass. Mass spectrometric and NMR analysis revealed that the in planta produced peptide differs from the synthetic version only in missing of N-terminal amidation. But its antimicrobial activity was in the range of the synthetic one. Taken together, we developed a protocol for plant-based production and purification of biologically active, hydrophobic and positively charged antimicrobial peptide.

Biobehavior in normal and tumor-bearing mice of tobacco mosaic virus.

Viral nanoparticles (VNPs) have shown great potential as platforms for biomedical applications. Before using VNPs for further biomedical applications, it is important to clarify their biological behavior in vivo, which is rare for rod-like VNPs. In this paper, a study of tobacco mosaic virus (TMV), a typical rod-like VNP, is performed on blood clearance kinetics, biodistributions in both normal and tumor-bearing mice, histopathology and cytotoxicity. TMV was radiolabeled with (125)I using Iodogen method for in vivo quantitative analysis and imaging purpose. In the normal mice, the accumulation of TMV in the immune system led to a rapid blood clearance. The uptake of TMVs in the liver was less than that in the spleen, which is opposite to the results observed in the case of spherical VNPs. No signs of overt toxicity were observed in examined tissues according to the results of histological analysis. In addition, similar biodistribution patterns were observed in U87MG tumor-bearing mice.

[Detection of tobacco mosaic virus (TMV) in Rehmannia glutinosa f. hueichingensis by IC-RT-PCR].

OBJECTIVE: To establish a rapid, sensitive and efficient detection method for tobacco mosaic virus (TMV), and provide technical support of TMV detection of Rehmannia glutinosa f. hueichingensis. The virus-free plantlets could be produced on a large scale to ameliorate breed degeneration caused by viral disease. METHOD: Specific primers were designed based on the conserved region of coat protein(CP) gene of TMV. Immunocapture RT-PCR (IC-RT-PCR) was employed to detect TMV and the sequence of the products was detected. RESULT: The expected nucleotide acid fragments were amplified by IC-RT-PCR. The homology of nucleotide acid sequence and amino acid sequence were 95.29% and 96.7% between the PCR products and the CP gene of TMV (accession number AY555269). CONCLUSION: The method was established for the detection of TMV in R. glutinosa f. hueichingensis by IC-RT-PCR. This detection combined molecular biology technology with immunology, was convenient for a quick, sensitive and simple detection of TMV.

The complete sequence of tobacco mosaic virus isolate Ohio V reveals a high accumulation of silent mutations in all open reading frames.

TMVOhioV was first described 1969 by [1] because it did break resistance of tomato breeding lines containing Tm-1- and Tm-2 resistance genes. It was obtained 1987 from Wetter (Saarbrücken, Germany) and transferred into the DSMZ-Plant Virus Collection (Braunschweig, Germany). A partial sequence of TMVOhioV, the CP gene, has been reported [11] and its comparison with a TMV type isolates (TMVtype), e.g. EMBL: V01409, revealed 50 point mutations in a total of 477 nucleotides (nts) leading to the replacement of only 7 amino acids (aa). In order to investigate the mutations in the non-translated regions and the number of silent mutation in the three other open reading frames (ORF), we sequenced the complete genome of isolate TMVOhioV and compared it to those of other Tobamoviruses.

Development of a one-step immunocapture real-time RT-PCR assay for detection of tobacco mosaic virus in soil.

Tobacco mosaic virus (TMV) causes significant losses in many economically important crops. Contaminated soils may play roles as reservoirs and sources of transmission for TMV. In this study we report the development of an immunocapture real-time RT-PCR (IC-real-time RT-PCR) assay for direct detection of TMV in soils without RNA isolation. A series of TMV infected leaf sap dilutions of 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (w/v, g/mL) were added to one gram of soil. The reactivity of DAS-ELISA and conventional RT-PCR was in the range of 1:102 and 1:103 dilution in TMV-infested soils, respectively. Meanwhile, the detection limit of IC-real-time RT-PCR sensitivity was up to 1:106 dilution. However, in plant sap infected by TMV, both IC-real-time RT-PCR and real-time RT-PCR were up to 1:106 dilution, DAS-ELISA could detect at least 1:103 dilution. IC-real-time RT-PCR method can use either plant sample extracts or cultivated soils, and show higher sensitivity than RT-PCR and DAS-ELISA for detection of TMV in soils. Therefore, the proposed IC-real-time RT-PCR assay provides an alternative for quick and very sensitive detection of TMV in soils, with the advantage of not requiring a concentration or RNA purification steps while still allowing detection of TMV for disease control.

Characterization of 10 tobacco vein banding mosaic virus isolates from China.

The complete RNA genome sequences of 10 tobacco vein banding mosaic virus (TVBMV) isolates from China were determined using five overlapping cDNA clones. TVBMV was divided into three groups for HC-Pro and CP and into two groups for P3 and 6K1. With more isolate sequences analyzed, the phylogenetic results suggest that TVBMV could be divided into four subgroups based on HC-Pro and CP and into three subgroups with P3 and 6K1. Nucleotide sequence diversity analysis showed geographical differentiation among the TVBMV isolates. Three of the 10 isolates were found to have undergone recombination and new recombination sites were identified in the TVBMV genome. All coding genes were under negative selection and the population appeared to have remained stable over a long period. This study also provides preliminary data on the 3´-untranslated region as potentially the best genome sequence for developing transgenic tobacco.

Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR.

AIMS: To develop a highly sensitive and rapid protocol for simultaneous detection and differentiation of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in pepper and tomato. In this study, we use the multiplex PCR technique to detect dual infection of these two viruses. METHODS AND RESULTS: A multiplex RT-PCR method consisting of one-tube reaction with two primer pairs targeted to replicase genes was developed to simultaneously detect TMV and ToMV in seed samples of pepper and tomato. Specific primers were designed from conserved regions of each of the virus genomes, and their specificity was confirmed by sequencing PCR products. RT-PCR detected up to 10(-6) dilution of total RNA extracted from infected leaves. Multiplex RT-PCR revealed the presence of both TMV and ToMV in three of 18 seed samples of tomato and one of 18 seed samples of pepper. CONCLUSIONS: The multiplex PCR assay was a cost effective, quick diagnostic technique, which was helpful in differentiating TMV and ToMV accurately. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex PCR assay described in this study is a valuable tool for plant pathology and basic research studies. This method may facilitate better recognition and distinction of TMV and ToMV in both pepper and tomato.

Status of tobacco viruses in Serbia and molecular characterization of tomato spotted wilt virus isolates.

In a four-year survey to determine the presence and distribution of viruses in tobacco crops at 17 localities of the Vojvodina Province and Central Serbia, 380 samples were collected and analyzed by DAS-ELISA. Out of the seven viruses tested, tomato spotted wilt virus (TSWV), potato virus Y (PVY), tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and alfalfa mosaic virus (AMV) were detected in 37.9, 33.4, 28.7, 23.9, and 15.5% of the total tested samples, respectively. TSWV was the most frequently found virus at the localities of Central Serbia, while PVY and CMV were the most frequent viruses in the Vojvodina Province. Single infections were prevalent in years 2005-2007 and the most frequent were those of PVY. A triple combination of those viruses was most frequent mixed infection type in 2008. The presence of all five detected viruses was confirmed in selected ELISA-positive samples by RT-PCR and sequencing. The comparisons of obtained virus isolate sequences with those available in NCBI, confirmed the authenticity of serologically detected viruses. Phylogenetic analysis based on partial nucleocapsid gene sequences revealed a joint clustering of Serbian, Bulgarian and Montenegrin TSWV isolates into one geographic subpopulation, which was distinct from the other subpopulation of TSWV isolates from the rest of the European countries. The high incidence of viruses in Serbian tobacco crops highlights the importance of enhancing farmers knowledge towards better implementation of control strategies for preventing serious losses.

Integration of biophysical photosynthetic parameters into one photochemical index for early detection of Tobacco Mosaic Virus infection in pepper plants.

Photosynthesis in host plants is significantly reduced by many virus families. The early detection of viral infection before the onset of visual symptoms in both directly and systemically infected leaves is critical in crop protection. Viral pathogens cause a variety of symptoms through modifications of chloroplast structure and function and the response of the photochemistry process is immediate. Therefore, chlorophyll fluorescence monitoring has been extensively investigated the last two decades as a tool for timely assessment of pathogenic threats. Alternatively, the analysis of Chla fluorescence transients offers several interlinked parameters which describe the fate of excitation energy round and through the photosystems. Additionally, OJIP fluorescence transients and leaf reflectance spectra methodologies serve for rapid screening of large number of samples. The objective of the present study was to achieve early detection of viral infection, integrating the multiparametric information of the Chla fluorescence transients and of the leaf reflectance spectra into one photochemical performance index. Infection decreased the maximum quantum yield of PSII (FV/FM), the effective quantum yield of PSII (ΦPSII), the CO2 assimilation rate (A) and the stomatal conductance (gs) in the studied TMV-pepper plant pathosystem, while non-photochemical quenching (NPQ) increased. Some parameters from the OJIP transients and the leaf reflectance spectra were significantly affected 24 h after infection, while others modified three to five days later. Similar results were obtained from systemically infected leaves but with one to three days hysteresis compared to inoculated leaves. Differences between healthy and infected leaves were marginal during the first 24 h post infection. The Integrated Biomarker Response tool was used to create a photochemical infection index (PINFI) which integrates the partial effects of infection on each fluorescence and reflectance index. The PINFI, which to the best of our knowledge is the first photochemical infection index created by the IBR method, discriminated reliably between the infected and healthy leaves of pepper plants from the first 24 h after infection with the TMV.

Live-cell imaging of viral RNA genomes using a Pumilio-based reporter.

We describe a method for localizing plant viral RNAs in vivo using Pumilio, an RNA-binding protein, coupled to bimolecular fluorescence complementation (BiFC). Two Pumilio homology domain (PUMHD) polypeptides, fused to either the N- or C-terminal halves of split mCitrine, were engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV). Binding of the PUMHDs to their target sites brought the split mCitrine halves into close proximity, allowing BiFC to occur and revealing the localization of viral RNA within infected cells. The bulk of the RNA was sequestered in characteristic inclusion bodies known as viral replication complexes (VRCs), with a second population of RNA localized in discrete particles distributed throughout the peripheral cytoplasm. Transfer of the TMV Pumilio recognition sequences into the genome of potato virus X (PVX) allowed the PVX RNA to be localized. Unlike TMV, the PVX RNA was concentrated in distinctive 'whorls' within the VRC. Optical sectioning of the PVX VRCs revealed that one of the viral movement proteins was localized to the centres of the RNA whorls, demonstrating significant partitioning of viral RNA and proteins within the VRC. The utility of Pumilio as a fluorescence-based reporter for viral RNA is discussed.

Rapid detection of tobacco mosaic virus using the reverse transcription loop-mediated isothermal amplification method.

Tobacco mosaic virus (TMV) can cause a severe disease that is capable of greatly reducing tobacco quality and yield. In this study, a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of TMV. The concentration of Mg(2+), reaction temperature and reaction time of the RT-LAMP were optimized to 5 mM, 65°C, and 60 min, respectively. The detection limit of the method was 100 times higher than that of RT-PCR. Visual inspection of RT-LAMP amplification demonstrated that positive and negative reactions exhibit distinctly different colours in daylight. Our results demonstrate that the method is stable, sensitive and specific.