Enhanced resistance and neutralization of defense responses by suppressors of RNA silencing

The effects of transgenic expression of the potato virus Y (PVY) HCPro silencing suppressor in tobacco were examined on infection by several viruses. Infection by tobacco mosaic virus (TMV) was reduced at 25 °C, but not at 33 °C. By contrast, systemic infection at 33 °C by the TMV expressing green fluorescent protein was promoted by the HCPro. Infection by tobacco rattle virus (TRV) was restricted to local necrotic lesions by the PVY HCPro. However, this resistance was neutralized by expression of the cucumber mosaic virus (CMV) 2b protein from TRV. By contrast, infection by either wild-type CMV or CMV with a deletion of the 2b gene was not affected. Similarly, infection by cauliflower mosaic virus, red clover necrotic mosaic virus (both limited to infection of the inoculated leaves of tobacco) or tomato bushy stunt virus (systemically infecting tobacco) was not altered by the expression of PVY HCPro. Therefore, it appeared that the PVY HCPro was able to induce defense responses at 25 °C, but not at 33 °C, where it actually neutralized a pre-existing defense response. Moreover, the CMV 2b protein was able to neutralize a defense response activated by HCPro in combination with TRV.     

Occurrence and distribution of ten viruses infecting cucurbit plants in Guilan province, Iran

During the 2006 and 2007 growing seasons, a systematic survey was conducted in open-field of melon (Cucumis melo L.), cucumber (C. sativus L.), squash (Cucurbita sp.), and watermelon (Citrulus lanatus L.) crops in 16 major cucurbit-growing areas of Guilan province in Iran. Symptomatic leaf samples were collected and screened by double-antibody sandwich ELISA (DAS-ELISA) or RT-PCR to detect Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Squash mosaic virus (SqMV), Papaya ringspot virus type W (PRSV-W), Watermelon chlorotic stunt virus (WmCSV), Melon necrotic spot virus (MNSV), Zucchini yellow fleck virus (ZYFV), and Ourmia melon virus (OuMV). The majority of tested samples (73.7%) were infected by at least one of the viruses considered. OuMV, ZYMV, WMV, and WmCSV were the most prevalent viruses and were detected in tested cucurbit plants. The incidence of multiple infections with 2 or more viruses was also relatively high, 63.3, 48.6, 42.7, and 26.7% of the infected samples of melon, cucumber, squash, and watermelon, respectively. The high incidence of OuMV and WmCSV suggested that these viruses might turn out to be an important threat for the melon and cucumber crops in the province. Key words: cucurbit viruses; incidence; DAS-ELISA; RT-PCR; multiple infection.     

Trapping of Tomato yellow leaf curl virus (TYLCV) and other plant viruses with a GroEL homologue from the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

Summary. To avoid destruction in the haemolymph of their vector, many plant circulative viruses interact with GroEL homologues produced by insect endosymbiotic bacteria. We have exploited this phenomenon to devise tools allowing trapping of plant viruses by either GroEL purified from the whitefly Bemisia tabaci or by whitefly GroEL over-expressed in E. coli. PCR tubes or 96-well plates coated with a GroEL preparation were incubated with cleared sap of virus infected plant leaves or insect vectors. GroEL-bound viruses were then identified by PCR or RT-PCR using virus-specific primers or by ELISA with virus specific antibodies. In this way Tomato yellow leaf curl virus (TYLCV) – a whitefly-transmitted geminivirus – was detected in plant sap, in extracts of leaf squashes and in homogenates of individual viruliferous whiteflies. Anti-GroEL antibody prevented TYLCV binding to GroEL. GroEL-bound virus was also detected by ELISA. GroEL was much more potent in binding TYLCV than commercial anti-TYLCV antibodies. In addition to several other geminiviruses, these procedures allowed detecting a variety of RNA viruses such as Cucumber mosaic virus (CMV), Prune dwarf virus (PDV) and Tomato spotted wilt (TSWV), but not Potato virus X and Potato virus Y (PVX and PVY), Grapevine leafroll-associated viruses (GLRV) and Tobacco mosaic virus (TMV). Predictions pertaining to viruses that do, or do not bind to GroEL, and applications in plant virus diagnosis, are presented.     

High prevalence of poleroviruses in field-grown pepper in Turkey and Tunisia

Open-field pepper crops were sampled in 2011 in Turkey and Tunisia and surveyed for the major pepper-infecting viruses. As expected, potato virus Y and cucumber mosaic virus (in both countries), and tobacco etch virus (in Turkey only) were quite frequent. However, poleroviruses were the most common viruses, with prevalences above 70 %. Partial sequence analyses revealed the occurrence of poleroviruses resembling either beet western yellows virus (BWYV) or pepper vein yellows virus in the sampled areas, with BWYV being predominant in Turkey but in the minority in Tunisia. Poleroviruses should therefore be taken into account in disease control of pepper crops in the Mediterranean area.     

Identification and sequence analysis of potyviruses infecting crops in Vietnam

Summary Fifty-two virus isolates from 13 distinct potyvirus species infecting crops in Vietnam were identified and the 3′ region of each genome was sequenced. The viruses were: bean common mosaic virus (BCMV), potato virus Y (PVY), sugarcane mosaic virus (SCMV), sorghum mosaic virus (SrMV), chilli veinal mottle virus (ChiVMV), zucchini yellow mosaic virus (ZYMV), leek yellow stripe virus (LYMV), shallot yellow stripe virus (SYSV), onion yellow dwarf virus (OYDV), turnip mosaic virus (TuMV), dasheen mosaic virus (DsMV), sweet potato feathery mottle virus (SPFMV) and a novel potyvirus infecting chilli, tentatively named chilli ringspot virus (ChiRSV). With the exception of BCMV and PVY, this is first report of these viruses in Vietnam. Further, rabbit bell (Crotalaria anagyroides) and typhonia (Typhonium trilobatum) were identified as new natural hosts of the peanut stunt virus (PStV) strain of BCMV and of DsMV, respectively. Sequence and phylogenetic analyses of the entire CP-coding region revealed considerable variability in BCMV, SCMV, PVY, ZYMV and DsMV. Correspondence: A/Prof. Rob M. Harding, Tropical Crops and Biocommodities Domain, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane 4001, Australia     

Immunochemical cross-reactivity between the dissociated capsid proteins of PVY group plant viruses

Primary (4–5-week) and long-term (12-week) antisera were elicited against the pyrrolidine-dissociated capsid proteins (D-proteins) of tobacco etch virus (TEV) and potato virus Y (PVY), and subsequently used to ascertain the degree of immunochemical cross-reactivity between several PVY group viral D-proteins. Primary antisera were highly specific for homologous antigen. However, immunodiffusion and cross-absorption experiments with long-term anti-TEV D-protein and anti-PVY D-protein sera revealed pronounced cross-reactivity among the D-proteins of 14 different PVY group viruses. Furthermore, all cross-reactions involved the same antibody population except in the case of celery mosaic virus (CeMV) D-protein where but a portion of the antibody population was reactive. Spur formation in immuno-diffusion experiments was only observed among heterologous D-proteins when they were either adjacent to homologous D-protein antigen or (in an opposite direction) when compared with CeMV D-protein. Wheat streak mosaic virus dissociated protein, in contrast, was not found to be cross-reactive with PVY group D-proteins in reciprocal tests, and no cross-reactions were observed with the dissociated proteins of 6 additional viruses outside the PVY group.     

Antiphytoviral activity of 2,4 dioxo hexahydro triazine.

Three treatments with 2,4 dioxo hexahydro triazine (DHT) significantly reduced the concentration of potato virus X (PVX) in systemically infected tobacco plants. In hypersensitive plants DHT caused a reduction in the number of local lesions produced by PVX. In systemic and hypersensitive hosts, treatment with DHT resulted in a more or less marked reduction in the concentration of, and in the number of local lesions caused by, potato virus Y (PVY), potato virus A (PVA), tobacco mosaic virus (TMV), and cucumber mosaic virus (CMV). Three days after treatment with DHT the incorporation of 32P into TMV RNA was significantly reduced, while that into host RNA was less affected.     

Development of a portable, high throughput biosensor system for rapid plant virus detection

Biosensors based on living cells are characterized by high sensitivity, selectivity and rapid response times. A novel portable cell biosensor system for the detection of plant viruses, based on immobilized ‘Vero’ cells carrying on their membrane virus specific antibodies was developed and was designated as High Throughput Bioelectric Recognition Assay (BERA-HTP). BERA-HTP was tested for the detection of purified Potato virus Y (PVY), Cucumber mosaic virus (CMV) and Tobacco rattle virus (TRV) and of CMV and TRV in single, as well as in mixed infections in two different plant host species. The sensor was based on live, mammalian cells, the membrane of which has been artificially saturated with antibodies specific to different plant viruses. The attachment of PVY, CMV or TRV viral particles to the homologous electroinserted antibodies caused a virus-specific change of the cell membrane electric potential that was not observed with virus-free samples or with heterologous viruses. Fluorescence microscopy observations showed that attachment of virus particles to the cell membrane bearing the homologous antibody, was associated with a decrease of [Ca²⁺]cyt. The perspective for the development of BERA-HTP as a portable, reliable and rapid (duration of assay for 96 samples: ∼70 min) detection method of plant viruses in the field is discussed.     

Quantitative assay for plant viruses using serologically specific electron microscopy

A rapid method for quantitative assay of plant viruses in crude extracts is described. The method involves counting virus particles attached to electron microscope grids that have been coated with specific viral antiserum. Dilutions of a crude extract of tobacco leaf infected with tobacco mosaic virus (TMV) gave 40 to 50 times more virus particles attached to grids specific for TMV than to those specific for potato virus Y (PVY). An extract of tobacco leaf infected with PVY gave approximately 20 times more virus particles attached to grids specific for PVY than to those specific for TMV. The log of the number of TMV particles attached to grids serologically specific for TMV decreased linearly with dilution of virus extract.     

Ultrastructure of cylindrical inclusions induced by viruses of the potato Y group as visualized by freeze-etching

Cylindrical inclusions induced by tobacco etch virus (TEV), potato virus Y (PVY), and turnip mosaic virus (TuMV) were examined by freeze-etch electron microscopy. The inclusion morphology in situ was similar to that previously reported from thin-sectioning studies, thus confirming that viruses in the potato Y group induce inclusions of characteristic morphology. Freeze-etching was also found to be useful for studying substructure. Inclusion subunits were clearly resolved when inclusions induced by TEV and PVY were examined both in situ and in isolated form.     

HC-Pro,a potyvirus RNA silencing suppressor,cancels cycling of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> plants

The mixed infection of Cucumber mosaic virus (CMV) and a potyvirus has been known to increase CMV titer in Nicotiana benthamiana plants, resulting in synergistic viral symptoms. We found that among three potyviruses—Potato virus Y (PVY), Turnip mosaic virus (TuMV), and Clover yellow vein virus (C1YVV)—synergistic effects on CMV (or a recombinant CMV vector) titers were most efficiently induced by a co-infection with PVY in N. benthamiana plants. In addition, the helper component-proteinase (HC-Pro) gene of PVY expressed by transgenic plants, which is a viral RNA silencing suppressor, was sufficient to cancel the cycling pattern of CMV titer, resulting in increased levels of overall CMV accumulation. Surprisingly, we found that the levels of CMV and the foreign protein expressed from the CMV vector were much higher in the HC-Pro-transgenic plants than the levels detected in the plants mixed-infected with CMV and PVY. The mechanism for canceling the cyclic infection of CMV by the HC-Pro protein alone is discussed in view of the interaction between RNA silencing and HC-Pro, as well as the possible involvement of the 3a protein.     

First report of cucumber mosaic virus subgroups i and ii on soybean, pea, and eggplant in iran

Cucumber mosaic virus (CMV) has the widest host range (> 1000 plant species) of any known plant virus, a?large number of vector species, and a?wide geographical distribution. A?survey was conducted to determine the incidence of CMV of subgroups I?and II on selected host crops in northern Iran. A?total of 935 leaf samples from 10 host crops (tomato, pea, tobacco, soybean, watermelon, broad bean, squash, cucumber, eggplant, and lettuce) showing virus disease-like symptoms were collected in 12 cities of Golestan and Mazandaran provinces (northern Iran) during 2009 and 2010. Among the field samples tested by double-antibody sandwich ELISA (DAS-ELISA), 275 samples were found to be infected by CMV. These were subsequently evaluated by compound ELISA with monoclonal antibodies. We found that 198 samples were infected by subgroup I, 98 samples by subgroup II and 45 samples by both virus subgroups. Twenty-four samples showed no reaction in compound ELISA. In presented paper, CMV subgroups I?and II (CMV-I?and CMV-II) have been reported for the first time on soybean, pea and eggplant in Iran, with subgroup I?being dominant in the north of the country. Keywords: CMV; subgroups I?and II; compound ELISA; monoclonal antibody.     

A virus-induced component of plant sap needed when aphids acquire potato virus Y from purified preparations

Aphids (Myzus persicae Sulz.) often transmitted potato virus Y (PVY) after probing through membranes into sap extracts made in solutions containing ethylenediametetraacetate (EDTA) and sodium diethyldithiocarbamate (Na-DIECA) but rarely did so when these chelating agents were omitted. Aphids did not transmit from purified virus preparations alone but did when they were first mixed with the supernatant obtained by ultracentrifuging a fresh sap extract of PVY-infected leaves; the supernatants alone were practically noninfective and aphids never transmitted virus from them. Aphids given separate access to supernatant and purified virus transmitted when they probed supernatant before virus but not when they probed virus first.Supernatants from extracts of PVY-infected leaves also helped aphids acquire henbane mosaic, tobacco severe etch and potato aucuba mosaic viruses.The results suggest that sap of plants infected with PVY contains some component, other than the virus particle, that is needed for virus acquisition by aphids probing through membranes.     

Towards a system for the identification and classification of potyviruses. II. Virus particle length, symptomatology, and cytopathology of six distinct viruses

The mean particle lengths of lettuce mosaic virus (LMV), bean common mosaic virus (BCMV), passionfruit woodiness virus (PWV), potato virus Y (PVY), sugarcane mosaic virus (SCMV), and one strain of bean yellow mosaic virus (BYMV) were all shown to be close to 750 nm. However, the length of two other serologically closely related strains of BYMV, variants of pea mosaic virus referred to as PMV and SPMV, had significantly longer particles, about 792 and 842 nm. Each of the distinct viruses had readily distinguishable host ranges. The strains of BYMV could also be distinguished from each other although they had many hosts in common with similar symptoms. Electron microscopy of plant cells infected by the viruses studied showed that the cell inclusions induced by infection were of two readily distinguishable types although some minor virus-specific differences were also observed. The BYMV strains and LMV induced long "relaxed" pinwheel inclusions whereas those induced by BCMV, PWV, PVY, and SCMV were shorter, "tighter," and curved. The significance of these observations to problems of potyvirus identification and classification is discussed.     

Ubiquitinated conjugates are found in preparations of several plant viruses

Recently D.D. Dunigan, R.G. Dietzgen, J.E. Schoelz, and M. Zaitlin (Virology 165, 310-312, 1988) demonstrated that a small proportion of the subunits of tobacco mosaic virus particles were conjugated with the small protein ubiquitin. We have now detected ubiquitinated conjugates in immunoblots of virion preparations of several other plant viruses, using anti-human ubiquitin antiserum. Based on their polyacrylamide gel migrations, plant virus-associated ubiquitin-immunoreactive proteins were considered to be possible virus structural protein-ubiquitin conjugates of the following viruses: barley stripe mosaic, brome mosaic, cowpea mosaic (two proteins), cowpea severe mosaic (two proteins), and satellite panicum mosaic. Ubiquitinated conjugates were not detected in immunoblots of preparations of cucumber mosaic virus and Cymbidium mosaic virus. The significance of ubiquitinated viral proteins remains to be determined.     

Independent assembly of virions in tobacco doubly infected by potato virus X and potato virus Y or tobacco mosaic virus

Enhancement of potato virus X (PVX) synthesis in tobacco cells doubly infected by PVX and potato virus Y (PVY) or by PVX and tobacco mosaic virus (TMV) is not a consequence of encapsidation of PVX-RNA by a protein coat containing the capsid subunits of PVY or TMV. Serology, electrophoresis, sedimentation analysis, and infectivity tests did not detect heterologous encapsidation of PVX-RNA, PVY-RNA, or TMV-RNA in doubly infected leaves.     

Characterization of some proteins associated with viruses in the potato Y group

Cytoplasmic inclusions induced by tobacco etch virus (TEV), potato virus Y (PVY), pepper mottle virus (PMV), bidens mottle virus (BiMV), and turnip mosaic virus (TuMV) as well as the respective viruses were purified from infected tissue and their constituent proteins investigated. A procedure is described for the simultaneous purification of virus and its associated inclusions. Electron microscopy of the purified inclusion preparations revealed structures characteristic of those seen in situ for each virus and are described as laminated aggregates shaped as triangles for TEV and as rectangles for BiMV, as scrolls for PVY and PMV, and as scrolls and laminated aggregates shaped as triangles for TuMV. Polyacrylamide electrophoresis of the cytoplasmic inclusions in 0.1% sodium dodecyl sulfate (SDS) revealed one protein component with an estimated molecular weight of 67,000 for PVY; 67,400 for PMV; 69,300 for BiMV; 69,600 for TEV; and 70,300 for TuMV. SDS electrophoresis of the viral coat proteins revealed anomalies, dependent in part on the history of the purified virus. Molecular weights of viral coat protein were estimated to be 26,000 and 32,000 for TEV; 28,000 and 34,000 for PVY; 28,000 and 34,000 for PMV; 28,000 and 33,000 for BiMV; and 27,000 and 36,000 for TuMV. Electrophoresis using different acrylamide gel concentrations and cellulose acetate support medium suggested that the larger of the two molecular weights estimated for each viral coat protein was a charge isomer.     

The tubule-forming NSm protein from Tomato spotted wilt virus complements cell-to-cell and long-distance movement of Tobacco mosaic virus hybrids

A Florida isolate of Tomato spotted wilt virus (TSWV) was able to complement cell-to-cell movement of a movement-defective Tobacco mosaic virus (TMV) vector expressing the jellyfish green fluorescent protein (GFP). To test for complementation of movement in the absence of other TSWV proteins, the open reading frame for the NSm protein was expressed from TMV constructs encoding only the TMV replicase proteins. NSm was expressed from either the coat protein or movement protein subgenomic promoter, creating virus hybrids that moved cell to cell in inoculated leaves of tobacco, providing the first functional demonstration that NSm is the TSWV movement protein. Furthermore, these CP-deficient hybrids moved into upper leaves of Nicotiana benthamiana, demonstrating that NSm can support long-distance movement of viral RNAs. Tubules, characteristic of the NSm protein, were also formed in tobacco protoplasts infected with the TMV-TSWV hybrids. The C-terminus of the NSm protein was shown to be required for movement. TMV-TSWV hybrids expressing NSm and GFP moved within inoculated leaves. Our combination of single-cell and intact plant experiments to examine multiple functions of a heterologous viral protein provides a generalized strategy with wider application to other viruses also lacking a reverse genetic system.