Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.Abbreviations CMV cucumber mosaiv virus - PBS phosphate buffered saline - TMV tobacco mosaic virus.     

Regulation of ADPglucose pyrophosphorylase in cucumber plants infected with the cucumber mosaic virus

ADPglucose pyrophosphorylase level and mechanisms regulating its activity were studied in cucumber plants infected with the cucumber mosaic virus at the stage of chronic infection. Studies carried out with partially purified preparations of the enzyme have shown that there was no substantial difference in the regulatory influence of the ratio 3-PGA/P₁, or in the number of binding sites of the effectors on the enzyme, but that the virus infection reduced the level of the enzyme in the tissues to 74% of the control and the 3-PGA/P₁ ratio to one half which resulted in a further decrease in ADPglucose pyrophosphorylase activity. In crude homogenate prepared from diseased plants, activity of the enzyme was reduced to 42% of the healthy control. The level of UDPglucose pyrophosphorylase was three times higher in cucumber leaf tissues than the level of ADPglucose pyrophosphorylase which was inhibited by both 3-PGA and P₁. Inhibitory effects of both these effectors were cumulated. The enzyme isolated from healthy plants was inhibited by inorganic phosphate more strongly than the enzyme isolated from diseased plants. UDPglucose pyrophosphorylase activity was increased in crude homogenate of diseased plants to 127% of the healthy control when the level of the enzyme was the same in the tissues of both healthy and diseased plants which was presumably connected with the enhanced rate of sucrose catabolism.     

Metabolic alterations in cotyledons of Cucurbita pepo infected by cucumber mosaic virus

Changes in the capacities of enzymes in various metabolic pathwayshave been measured during infection of cotyledons of Cucurbitapepo L. with cucumber mosaic virus (CMV). Starch accumulationand low sucrose content, which are characteristic of the earlystages of infection, are reversed in the later stages of infection.The decline in starch correlated with a reduced capacity forstarch synthesis (ADP-glucose pyrophosphorylase) and a risein the capacity for starch degradation (total starch hydrolase,starch phosphorylase). ¹⁴CO₂ feeding experiments, conductedat saturating CO₂ concentration, show that the newly-assimilatedcarbon was lost at a lower rate from infected cotyledons andless was incorporated into structural carbohydrates, phosphorylatedintermediates plus organic acids, more into soluble sugars,amino acids and proteins. At a later stage of infection therewere dramatic increases in respiratory capacity and a substantialalteration of carbohydrate metabolism. The infection had a largestimulatory effect on the capacity for oxidative pentose-phosphatepathway (glucose-6-phosphate dehydrogenase, 6-phospho-gluconatedehydrogenase), glycolysis (ATP- and pyrophosphate-dependentphosphofructokinases), tri-carboxylic acid cycle (isocitratedehydrogenase, fumarate hydratase), anaplerotic reactions (NAD-dependentmalic enzyme, phosphoenolpyruvate car-boxylase) and oxidativeelectron transport (cytochrome c oxidase). While there wereno overall changes in photosynthetic rate (measured in saturatingCO₂), infection either reduced (Rubisco and glycerate kinase)or did not affect (chloroplastic fructose bis-phosphatase andhydroxypyruvate kinase) the capacities of the photosyntheticcarbon reduction pathway or the photosynthetic carbon oxidationpathway. Key words: Plant-virus interaction, sucrose, starch, enzymes, ¹⁴CO₂ incorporation, O₂ flux     

Response of tomato plants infected with cucumber mosaic virus to foliar sprays of gibberellic acid

The effect of spraying tomato plants infected with cucumbermosaic virus (CMV) with gibberellic acid (GA) 1 and 5 ppm hasbeen studied in a glasshouse. Measurements showed that the differencein size between GA-treated healthy and infected plants eitherdisappeared or became insignificant and this was true at bothhigh and low planes of nutrition. Virus content was not reducedby GA. Virusinduced checks to leaf production and stem elongationtended to disappear in unsprayed plants but leaf area and shootweight did not show natural recovery. The stimulating effectof GA was primarily on leaf expansion and secondarily on shootweight.     

Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02-0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.     

Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02–0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.     

Mild mosaic of spiraea caused by cucumber mosaic virus

A disease of spiraea(Spiraea xvanhouttei) manifested in leaves by very mild, mostly hardly perceptible mosaic, was found to be caused by cucumber mosaic virus (CMV) infection. The proof was given on the basis of responce of differential plants after virus transmission, by immunosorbent electron microscopy and ELISA.     

Temperature-related effects of treatments with jasmonic and salicylic acids on Arabidopsis infected with cucumber mosaic virus

Plant-virus interactions are affected by environmental factors, including temperature. Plant defenses are often inhibited by high or low temperature. In this study, oxidative damage and gene expression were detected in Arabidopsis thaliana infected with cucumber mosaic virus (CMV) at different temperatures. Before virus inoculation, plants were treated with jasmonic acid (JA) and salicylic acid (SA), both of which are important signaling molecules in plant defense responses. The levels of MDA and hydrogen peroxide (H₂O₂), and electrolyte leakage were significantly higher in CMV-infected leaves at 15 and 37°C. The accumulation of H₂O₂ and superoxide radical (O ₂ ^·? ) was obviously suppressed by spraying with JA followed by SA (JA → SA) at different temperatures. The CMV-CP expression analysis showed that virus replication was inhibited efficiently in the (JA → SA) treatment. Therefore, many JA- and SA-responsible resistance genes were quantified; MPK4 was expressed highly and steadily in the (JA → SA) treatment. To further confirm the role of MPK4, the CMV-CP gene expression was evaluated in wild-type Arabidopsis and its mpk4 mutant infected with CMV. The results suggested that MPK4 might play an important role in the antagonism between JA and SA at temperature fluctuation.     

High temperature inactivation of cucumber and alfalfa mosaic viruses in Nicotiana rustica cultures

Cucumber mosaic (CMV) and alfalfa mosaic (AlfMV) viruses could not be detected in Nicotiana rustica tissues cultured at 32 °C for 16–18 days or at 40 °C for 5 days, but infectivity remained high in comparable tissue cultured at 22 °C. Incubation of infected cultures at 28–30 °C resulted in an initial reduction followed by a partial recovery in the infectivity of both viruses. The infectivity of CMV in tissues grown between 12 and 32 °C was highest in cultures grown at 12 °C. Although CMV infectivity was not detected in cultures after 16–18 days at 32 °C, virus was eliminated only after a further 30 days at 32 °C. When cultures were transferred from 32 to 22 °C after shorter treatment periods, infectivity rapidly increased to levels higher than those of infected tissues grown continuously at 22 °C. At 40 °C, CMV was eliminated from infected tissues after 9 days and AlfMV after 7 days. Cultures grown continuously at 40 °C deteriorated rapidly but, when grown under diurnal alternating periods of 8 h at 40 °C and 16 h at 22 °C, they remained viable and CMV was also inactivated.     

Isolates of cucumber mosaic virus from spontaneously infected plants ofChelidonium majus andImpatiens parviflora

Cucumber mosaic virus (CMV) isolates obtained from spontaneously infected (1)Chelidonium majus L. and (2)Impatiens parviflora DC. plants growing in the same part of a scree forest in central Bohemia showed differences in ten test plants, but not in further 26 plant species tested and in TIP. Slight quantitative differences between both isolates were obtained in serological tests. Properties of both isolates did not suit precisely any proposed CMV strain classification.     

Detection of cucumber mosaic virus in some ornamental plants and elimination of nonspecific ELISA reactions

During surveys carried out in 2008 in the nurseries of some ornamental and medical plants, about 90 plant samples belonging to six plant species were collected. Cucumber mosaic virus (CMV) was detected by routinely double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) in most tested plants. In Vinca rosea, Ocimum basilicum and Pelargonium sp., which reacted positively for CMV, 100% of the samples were infected. High absorbance values were obtained when extracts were prepared from these species and then examined by DAS-ELISA. The results indicated that CMV concentration on O. basilicum is greater than 50 μg ml^?1 when compared with purified CMV standards. High absorbance values occur even under conditions fully unsuitable for the detection of antigens. This result suggested that the strong ELISA absorbance values were nonspecific reactions with materials in plant extracts. So, other detection methods including dot blot, Western blot and bioassay was used for comparing with ELISA test. The nonspecific reactions were substantiated when CMV was not detected by dot blot and Western blot. Also, infectious CMV was not detected in bioassay involving inoculation of extracts onto Chenopodium amaranticolor and Nicotiana tabacum var. White Burley plants as indicator hosts. Addition of bovine serum albumin (BSA) or polyvinylpyrolidene (PVP) to extraction buffer or to wells after IgG coating for DAS-ELISA reduced nonspecific reactions. Finally, CMV was isolated from V. rosea symptomatic plants, which give a positive reaction by DAS-ELISA, dot blot, Western blot and bioassay. CMV vinca-isolate was identified based on transmission, host range, serological tests, purification and electron microscope.     

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses — cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.     

Determining the relative roles of different aphid species as vectors of cucumber mosaic and bean yellow mosaic viruses in lupins

Glasshouse and field studies were done to determine the relative roles of different colonising and non-colonising aphid species as vectors of two non-persistently transmitted viruses, cucumber mosaic cucumovirus (CMV) and bean yellow mosaic potyvirus (BYMV) in narrow-leafed lupin (Lupinus angustifolius) crops in Australia. The abilities of nine different aphid species in transmitting CMV from infected to healthy lupins and BYMV from infected subterranean clover to healthy lupins were compared in the glasshouse using 5–10 min acquisition access feeds. The percentage transmission efficiencies found with lupin-colonising aphid species were (CMV/BYMV): Acyrthosiphon kondoi (6/15), Aphis craccivora (10/14) and Myzus persicae (11/77). With non-colonising species the respective efficiencies were: Brachycaudus rumexicolens (0.9/0), Lipaphis erysimi (4/8), Rhopalosiphum maidis (9/6), R. padi (5/5), Sitobion miscanthi (2/11) and Therioaphis trifolii (4/5). When flying aphids were trapped in the field in four successive years (1993–1996) on vertical nets downwind of virus-infected lupins, 13 different species were caught at a “wheatbelt” site and 18 at an urban irrigated site. Of 2833 aphids caught at the “wheatbelt” site, 64 transmitted CMV to lupin test plants. At the irrigated site, numbers of aphids transmitting CMV/numbers caught were 12/186 while the corresponding numbers for BYMV were 11/727. M. persicae, A. kondoi and R. padi transmitted both viruses, while additional vectors of CMV found were A. craccivora, Acyrthosiphon pisum, B. rumexicolens, L erysimi, R. insertum, T. trifolii and Toxoptera citricidus. Averaged over four years, A. kondoi accounted for 50% of CMV transmissions at the “wheatbelt” site, M. persicae for 16% and R. padi for 22%, and these three species were caught in the greatest numbers, comprising 28%, 13% and 37% respectively of the total catch. At the irrigated site R. padi accounted for half the CMV transmissions, while R. padi and A. kondoi together accounted for most of the BYMV transmissions. R. padi, A. kondoi, M. persicae and T. citridus were the most common aphid species at this site. These findings suggest that M. persicae, A. kondoi and R. padi are the aphid species likely to be most important as vectors of CMV and BYMV in narrow-leafed lupins grown in mediterranean-type climatic zones of southern Australia.     

The effect of 2,4-dichlorophenoxyacetic acid on the metabolic utilization of free carbohydrates in cucumber mosaic virus infected cucumber plants

The application of 2,4-dichlorophenoxyacetic acid at a concentration of 1.0 × 10⁻³ M toCucumis sativus brings about a decrease in the activity of carbohydrate catabolizing enzymes in the leaves of experimental plants. On the contrary, in CMV infected plants the activity of sucrase, glucokinase, fructokinase, phosphoglucoisomerase and glucose-6-phosphate dehydrogenase are enhanced at the same time. The application of 2,4-D to virus infected plants promotes this effect further, so that the activities of the enzymes investigated are twice as high as those in the control.     

Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.