Production and properties of monoclonal antibodies to Solanum nodiflorum mottle sobemovirus

Black raspberry necrosis virus (BRNV) reaches only very low concentrations in herbaceous plants and is difficult to maintain in culture. However, in a mixed culture with an unrelated virus, Solanum nodiflorum mottle (SNMV), in the genus Sobemovirus, the concentration of BRNV particles increases about 1000‐fold. In attempts to produce monoclonal antibodies (MAbs) to BRNV for diagnostic use, purified virus particles from the mixed virus culture were used as immunogen and the resultant antibodies screened against cultures of SNMV alone, BRNV+SNMV and healthy plant extracts. None of the virus‐specific MAbs obtained in this way was specific to BRNV but six were specific to SNMV. Although the original objective was not achieved, the SNMV MAbs were characterised and used to study serological properties of SNMV and other Sobemoviruses. Characterisation of the six SNMV MAbs showed that four were IgG3, one IgG1 and the other IgG2b. SNMV was detected by all six MAbs in ELISA, by five in Western blotting, by three in agarose gel double diffusion tests, but only one was suitable for trapping virus particles in immuno‐electron microscopy (IEM). In Western blotting using virus in sap extracts of Nicotiana clevelandii, each of the five MAbs detected a single major band of Mc. 31 000 in sap containing SNMV, and additional bands of lower mass attributed to degradation of coat protein. In various serological tests, no cross‐reactions were detected between SNMV and seven other viruses from the genus Sobemovirus. However, in IEM but not in Western blotting, significant cross‐reactions were observed between SNMV and Velvet tobacco mottle virus, another species from the genus Sobemovirus. The significance of these different findings is discussed.     

Non-propagative translocation of velvet tobacco mottle virus in the mirid, Cyrtopeltis nicotianae

Nymphs of the mirid, Cyrtopeltis nicotianae became infective when injected with velvet tobacco mottle virus (VTMoV). Injections of amounts between 1 and 154 ng into the haemocoele induced 2/60 to infect test plants and these two nymphs contained 50 and 63 ng of virus respectively. Injection of amounts between 15 and 2400 ng rendered 11/47 nymphs infective. This observation is characteristic of a circulative association. However, there is no evidence that the salivary glands are involved in transmission and the virus is therefore defined as translocating, rather than circulating, in the mirid vector. Mirids which acquired infectivity by feeding lost it between 5 and 9 days after completion of acquisition, and the most rapid loss of infectivity occurred within 2 days. Nine days after acquisition none contained antigen detectable by ELISA, but detectable antigen decreased less rapidly than infectivity, and at all times more mirids contained antigen than were able to transmit. Mirids containing antigen carried between 150 and 3340 ng each. Thus, although VTMoV can be transmitted by its mirid vector following introduction of virus into the body cavity by injection, VTMoV is not propagative. Nor does the presence of virus within the mirid guarantee an ability to transmit.     

天津近郊番茄病毒类型的研究

应用ELISA间接法检测出天津近郊番茄上至少有9种病毒类型。且首次检测出国内在番茄上尚未报道的番茄黑环病毒(TBRV)、绒毛烟斑驳病毒(VTMoV)。经生物学测定还分离出番茄上表现枯斑型的86-125和在豇豆上产生大红斑的86-95-15均与13种抗血清呈阴性反应的杆状待测病毒。     

Molecular Characterization and Distribution of Cucumber green mottle mosaic virus in China

Cucumber green mottle mosaic virus (CGMMV) is an emerging virus on watermelon in China. We report here the almost complete nucleotide sequence and the characterization of the genome of a Chinese isolate (CGMMV-LN). Nucleotide sequence comparisons showed CGMMV-LN is closely related to CGMMV-KOM, with 99.4% identity. On the basis of the nucleotide sequence, a digoxigenin-labelled cDNA probe CG, complementary to the replicase gene region of CGMMV was synthesized. The specificity and sensitivity of the probe was tested. The detection limit of the method was equivalent to 0.8 μg fresh tissues infected by CGMMV. Two hundreds and eighteen watermelon samples collected from different regions in China during 2006–2007 were tested by this method. The distribution pattern of CGMMV in China during these years was revealed. The virus has spread in five provinces of China so far, including Liaoning, Hebei, Guangdong, Hubei and Shandong and might be an increasing tendency, which provides important information for CGMMV control in China.     

Resistance in rice to Rice yellow mottle virus and evidence of non-seed transmission

Nine rice cultivars were evaluated under screenhouse conditions for resistance to Rice yellow mottle virus (RYMV) and possible seed transmission. Completely randomised design with three replications was used. In Experiment 1, the seedlings were inoculated with the virus at two weeks after planting. In Experiment 2, the seeds collected from Experiment 1 were dried for four weeks before planting. For each genotype, the seeds from healthy plants were planted as a control. Disease incidence and severity (scales 1–9), yield and yield components were recorded. Statistical analyses included Area Under the Disease Progress Curve (AUDPC) and independent t test. The cultivars FARO 37, FARO 52 and Gigante were highly resistant, whereas WAB189-B38HB was resistant. Paddy yield was highest (3.6 g) in FARO 37. There were no symptoms of virus disease in all the plants originating from the seeds of RYMV-infected plants. The differences between the seeds from infected and healthy plants for all the measured traits were not significant (p > 0.05). The number of days to seedling emergence was uniform (5.5 days) in all the cultivars. Plant height, number of tillers per plant, number of days to heading and paddy yield from the seeds of virus-infected plants varied from 54.8 to 68.4 cm, 17 to 21, 85.3 to 96 days and 2.7 to 4 g, respectively. Conversely, a range of 54.9–68.7 cm, 17–22, 83–95 days and 2.8–4.1 g was found in the seeds of healthy plants. Selection and cultivation of high-yielding, resistant and healthy seeds would enhance food security.     

Agrobacterium-mediated transformation of monocot and dicot plants using the NCR promoter derived from soybean chlorotic mottle virus

The NCR promoter (PNCR) from soybean chlorotic mottle virus (SoyCMV) was used to express the selectable marker, neomycin phosphotransferase (nptII) gene, in Agrobacterium-mediated transformation of both monocot (rice) and dicot (tobacco) plants. A multi-cloning site for insertion of a gene of interest into the binary vector pTN is located proximal to the right border region of T-DNA. When chimeric genes under the control of other strong promoters were located in a head-to-head orientation to the PNCR-nptII gene, kanamycin-resistant tobacco shoots were generated more efficiently than when using the original pTN vectors. This suggests that the enhancer-like sequences in the promoters adjacent to PNCR may promote expression of the PNCR-nptII gene. Received: 20 August 1999 / Revision received: 16 November 1999 / Accepted: 19 November 1999     

Differences of reconstitution process between tobacco mosaic virus and cucumber green mottle mosaic virus by synchrotron small angle X-ray scattering using low-temperature quenching

The differences of the reconstitution process of tobacco mosaic virus (TMV) and its mutant, cucumber green mottle mosaic virus (CGMMV) were investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. The reconstitution in an aqueous solution is completely stopped below 5°C. The TMV and CGMMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5°C on a series of solutions prepared by low-temperature quenching after incubation at 20°C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. The incubation of RNA and protein of CGMMV did not reconstitute at the initial reaction stages below 5 min and then began to reconstitute gradually. After 60 min, the radius of gyration for CGMMV reconstitution process reached almost the value for the initial stage of TMV reconstitution process. This is due to the fact the formation of double-layered disk in CGMMV protein is much slower than in TMV protein.     

Purification and some properties of strawberry mottle virus

Strawberry mottle virus (SMoV) (three isolates: HJ, 3E and N) were transmitted to Chenopodium quinoa plants by sap inoculation. All three isolates induced very similar symptoms consisting of chlorotic spots and ringspots in inoculated leaves, and vein chlorosis, mottling, and dwarfing of the upper leaves. SMoV isolate HJ was purified from infected C. quinoa by homogenisation with 10 mM phosphate buffer, pH 7.2 containing 5% Triton X-100, followed by differential, sucrose density-gradient and CsCl equilibrium density-gradient centrifugations. A fraction with a buoyant density of 1.42g^- cm^-3 after CsCl density-gradient centrifugation was highly infectious to C. quinoa and contained many isometric virus-like particles c. 37 nm in diameter. These virus-like particles were never found in fractions from uninfected preparations. Electrophoretic analysis of a fraction containing virus-like particles revealed that these particles might have a single coat protein subunit with the apparent molecular mass of 26 K daltons and one nucleic acid of 6.6 kilobases. Double-stranded RNA analysis of isolate HJ-infected or uninfected C. quinoa and Fragaria vesca var. semperflorens seedling line ‘Alpine’ plants showed that both infected plants had two infection-specific dsRNA bands of mol. wts 4.5 and 3.9 × 10⁶.     

The Commelina yellow mottle virus promoter drives companion-cell-specific gene expression in multiple organs of transgenic tobacco

Summary.  Previous work has demonstrated that some endogenous plant gene promoters are active in selective companion cells of the phloem, depending on organ types and developmental stages. Here we report that the Commelina yellow mottle virus (CoYMV) promoter is active in the companion cells of leaves, stems and roots of transgenic Nicotiana tabacum cv. Xanthi NN, using β-glucuronidase (GUS) as a reporter. Thus, the CoYMV promoter has a broad organ specificity. This promoter can be useful in molecular studies on the functions of companion cells in many aspects of phloem biology, such as regulation of long-distance transport, macromolecular traffic, plant development and interaction with pathogens. It may also be useful in engineering crops that produce specific gene products in the companion cells to block long-distance movement of pathogens. Received February 5, 2002; accepted March 27, 2002; published online July 4, 2002 RID="*" ID="*" Correspondence and reprints: Department of Plant Biology and Plant Biotechnology Center, 207 Rightmire Hall, Ohio State University, 1060 Carmack Road, Columbus, OH 43210, U.S.A.     

Production of Antiserum to Recombinant Coat Protein for Detecting Lily mottle virus in Yunnan, China

Lily mottle virus (LMoV), Potyvirus genus, is very difficult to purify for the preparation of diagnostic antisera. The coat protein (CP) gene of LMoV was amplified by RT-PCR from infected plants and cloned into the prokaryotic pET-30a vector to generate the recombinant plasmid pET-CP. The resulting carboxy-terminal His-tagged CP was over-expressed in Escherichia coli BL 21 cells by Isopropyl-β- d -thiogalactoside (IPTG) induction and purified over Ni-NTA affinity columns. The purified CP was used to elicit a polyclonal antiserum in rabbits. The antiserum had a titre of 1 : 128 in double diffusion tests, and specifically recognized LMoV in Western blots, Enzyme-Linked Immunosorbent Assays and Immuno-Electron Microscopy. The CP antiserum was also used to evaluate LMoV infection of lily bulbs used for commercial production in Yunnan, China. Substantial levels of infection were found in both imported and native bulbs, and provide the basis to implement flexible, rapid and large-scale virus indexing of lily plants for use in propagation and to meet virus-free quarantine regulations.     

Delivery of macromolecules to plant parasitic nematodes using a tobacco rattle virus vector

Plant parasitic nematodes cause significant damage to crops on a worldwide scale. These nematodes are often soil dwelling but rely on plants for food and to sustain them during reproduction. Complex interactions occur between plants and nematodes during the nematode life cycle with plant roots developing specialized feeding structures through which nematodes withdraw nutrients. Here we describe a novel method for delivering macromolecules to feeding nematodes using a virus-based vector [tobacco rattle virus (TRV)]. We show that the parasitic nematode Heterodera schachtii will ingest fluorescent proteins transiently expressed in plant roots infected with a TRV construct carrying the appropriate protein sequence. A prerequisite for this delivery is the presence of replicating virus in root tips prior to the formation of nematode-induced syncytia. We show also that TRV vectors expressing nematode gene sequences can be used to induce RNAi in the feeding nematodes.     

Occurrence of symptomless source of Piper yellow mottle virus in black pepper (Piper nigrum L.) varieties and a wild Piper species

Symptomless nature of Piper yellow mottle virus (PYMoV) infection in three varieties of black pepper (Piper nigrum) (Panniyur 1, Panniyur 5 and Panchami) and a wild species of Piper (Piper colubrinum) was confirmed by polymerase chain reaction (PCR) using PYMoV specific primers. The virus could be transmitted from these PYMoV-infected symptomless plants onto symptom producing black pepper cv. Karimunda through mealybug vector, Ferrisia virgata and by graft transmission. About 20–50% seedlings showed typical symptoms of the PYMoV at 30 days after mealybug inoculations while it was 75–94% at 90 days after inoculation. PCR test of the inoculated seedlings confirmed the presence of PYMoV in 50–64%, 76–100% and 80–100% of plants in 30, 60 and 90 days after inoculation, respectively. Similarly, 50–66%, 91–100% and 100% of graft-transmitted plants showed typical symptoms of the disease at 30, 60 and 90 days after grafting. PCR test of the graft-transmitted plants showed 100% PYMoV infection at 60 days after grafting. The results clearly demonstrated the existence of PYMoV-infected symptomless plants that can act as source for secondary spread of the virus in the field.     

Localization of Banana bunchy top virus and cellular compartments in gut and salivary gland tissues of the aphid vector Pentalonia nigronervosa

Banana bunchy top virus (BBTV) (Nanoviridae: Babuvirus) is transmitted by aphids of the genus Pentalonia in a circulative manner. The cellular mechanisms by which BBTV translocates from the anterior midgut to the salivary gland epithelial tissues are not understood. Here, we used multiple fluorescent markers to study the distribution and the cellular localization of early and late endosomes, macropinosomes, lysosomes, microtubules, actin filaments, and lipid raft subdomains in the gut and principal salivary glands of Pentalonia nigronervosa. We applied colabeling assays, to colocalize BBTV viral particles with these cellular compartments and structures. Our results suggest that multiple potential cellular processes, including clathrin‐ and caveolae‐mediated endocytosis and lipid rafts, may not be involved in BBTV internalization.     

Genetic basis and mapping of the resistance to rice yellow mottle virus. I. QTLs identification and relationship between resistance and plant morphology

 Rice yellow mottle virus (RYMV) resistance QTLs were mapped in a doubled-haploid population of rice, ‘IR64/Azucena’. Disease impact on plant morphology and development, expression of symptoms and virus content were evaluated in field conditions. Virus content was also assessed in a growth chamber. RYMV resistance was found to be under a polygenic determinism, and 15 QTLs were detected on seven chromosomal fragments. For all of the resistance QTLs detected, the favourable allele was provided by the resistant parent ‘Azucena’. Three regions were determined using different resistance parameters and in two environments. On chromosome 12, a QTL of resistance that had already been detected in this population and another indica/japonica population was confirmed both in the field and under controlled conditions. Significant correlations were observed between resistance and tillering ability, as measured on control non-inoculated plants. In addition, the three genomic fragments involved in resistance were also involved in plant architecture and development. In particular, the semi-dwarfing gene sd-1, on chromosome 1, provided by the susceptible parent, ‘IR64’, mapped in a region where resistance QTLs were detected with most of the resistance parameters. In contrast, the QTL of resistance mapped on chromosome 12 was found to be independent of plant morphology. Received: 20 April 1998 / Accepted: 30 April 1998     

Efficacy of pymetrozine against Myzus persicae and in reducing potato virus Y transmission on tobacco plants

Forty‐four parthenogenetic lineages of Myzus persicae s.l. (Sulzer) from tobacco crops and peach orchards located in various regions of Greece were examined to determine their response to the insecticide pymetrozine using leaf‐dip bio‐assays. The results show that the aphid has not developed resistance, as all lineages exhibited resistance factors bellow 6.0. In transmission experiments of potato virus Y (PVY) using a lineage of the tobacco‐adapted subspecies M. persicae nicotianae Blackman on tobacco plants, one foliar application with pymetrozine provided adequate protection for 7 days. Pymetrozine significantly reduced both virus acquisition and inoculation compared with the untreated control and the reduction was comparable to a mineral oil application. These results are discussed in terms of the advantage of incorporating pymetrozine as a compound of pest management strategies against M. persicae s.l. and for control of non‐persistent viruses, especially in crops such as tobacco because of the high selection pressure from neonicotinoids resulting in potential of resistance developing in aphid populations.     

Proteome analysis of cultivar-specific deregulations of Oryza sativa indica and O. sativa japonica cellular suspensions undergoing rice yellow mottle virus infection

We have used two-dimensional gel electrophoresis with mass spectrometry analysis to study the temporal patterns of protein expression during RYMV (Rice yellow mottle virus) infection in rice cells of two cultivars: IR64, Oryza sativa indica, susceptible, and Azucena, O. sativa japonica, partially resistant to RYMV. Proteomic analysis of nonstressed and RYMV inoculated cells showed statistically significant changes in the relative levels of 40 IR64 proteins and 24 Azucena proteins. Protein identification using mass spectrometry was attempted for all the differentially regulated proteins. This global analysis detected 32 hypothetical "new" proteins. Nineteen differentially regulated proteins were identified for IR64 cultivar, while 13 were identified for Azucena cultivar, including proteins in three functional categories: metabolism, stress-related proteins, and translation. These data revealed that a number of proteins regulated by abiotic stress response pathway were activated by RYMV in both cultivars (such as salt-induced protein, heat shock proteins (HSPs), superoxide dismutase (SOD), and others have functions consistent with the susceptibility or partially resistance trait (such as dehydrin, proteins involved in glycolysis pathway).     

Comparison of ELISA and immunoblotting techniques for the detection of cherry mottle leaf virus

Formaldehyde treated cherry mottle leaf virus (ChMLV) and the isolated coat protein were used successfully for the production of polyclonal and monoclonal antibodies. The monoclonal antibodies had a titre of 1:51 200 and consisted of IgG1 and IgG2. The antibodies reacted with all 11 isolates of ChMLV, from five locations in Canada and the USA, included in this study. Several serological procedures were assessed to compare their sensitivity for detecting ChMLV. Plate-trapped antigen ELISA (PTA-ELISA) and dot-blot immunobinding assay (DBIA), using virus specific MAbs, were the most sensitive tests in this study. Triple antibody sandwich ELISA (TAS-ELISA) and Western blot were found to be less sensitive. Dilution of the samples appeared to increase the sensitivity of both PTA-ELISA and Western blot detection. Young leaves and flowers of Prunus avium were the best tissue for detecting the virus which could also be detected in the fruit and leaves of P. tomentosa. April and May were optimal for detection of the virus in the field, whereas both April to May and August to September were optimal for screenhouse-grown plants.     

Immunodiagnosis of Parietaria mottle virus in Tomato Crops Using a Polyclonal Antiserum against its Coat Protein Expressed in a Bacterial System

Recombinant DNA technology was used to raise a polyclonal antiserum against the coat protein (CP) of Parietaria mottle virus (PMoV). The CP gene was expressed in Escherichia coli as a fusion to a 6xHis tag and purified by affinity chromatography. Recombinant purified protein was used as antigen to raise a polyclonal antiserum. This polyclonal antiserum consistently detected PMoV specifically infected tomato plants from different commercial tomato crops by indirect enzyme-linked immunosorbent assay (I-ELISA) and direct tissue-printing immunoassay (DTBIA).     

States of aggregation of the dahlemense strain of tobacco mosaic virus protein and their relation to crystal formation.

The aggregation of the protein of the dahlemense strain of tobacco mosaic virus has been studied by electron microscopy and ultracentrifugation. The aggregates formed are similar to those formed by the vulgare strain, although the particular conditions for their formation are often rather different. Helix formation by dialysis of A protein at pH 8 to acid pH is much more efficient if an intermediate step at pH 7 is introduced. The 20 S particle or two-layer disk is stable over a wide range of pH and ionic strength values. There is no tendency to form short stacks of disks at high ionic strength; instead, 30 S particles are formed that correspond to a pair of interlocked disks giving a “figure-of-eight” appearance in electron micrographs. These particles appear to be the “building blocks” of the protein crystal.