Alteration in carbon partitioning induced by the movement protein of tobacco mosaic virus originates in the mesophyll and is independent of change in the plasmodesmal size exclusion limit

The influence of the 30 kDa movement protein of tobacco mosaic virus (TMV-MP) on carbon partitioning in trans-genie tobacco plants (Nicotiana tabacum cv. Xanthi) expressing the TMV-MP was investigated. Using reciprocal grafting of transgenic tobacco plants expressing this movement protein and vector control plants, as well as transgenic tobacco plants expressing the TMV-MP in phloem cells only, we showed that the interactive site involved in carbon allocation to roots is localized to the mesophyll tissue. Biomass partitioning experiments conducted on transgenic plants, in which various deletion mutant forms of the TMV-MP (two of which included deletions in the domain responsible for increasing the size exclusion limit) were expressed, revealed that the TMV-MP exerts its influence on carbon allocation via a mechanism that is completely independent of the TMV-MP-induced increase in the plasmodesmal size exclusion limit. Furthermore, small N- and C-terminal deletions in the MP revealed the complexity of the interactions likely to be involved between the MP and an endogenous regulatory mechanism. We propose that the TMV-MP interferes with an endogenous signal transduction pathway that involves macromolecular trafficking through plasmodesmata to regulate biomass partitioning between the source and various sink tissues.     

Cucumber mosaic virus infection affects sugar transport in melon plants

Viral infection often affects carbon assimilation and metabolism in host plants. To better understand the effect of cucumber mosaic virus (CMV) infection on sugar transport, carbohydrate levels and the amounts of the various sugars in the phloem sap were determined in infected melon (Cucumis melo L.) plants. Source leaves infected with CMV were characterized by high concentrations of reducing sugars and relatively low starch levels. The altered level of carbohydrates was accompanied by increased respiration and decreased net photosynthetic rates in the infected leaves. Although stachyose was the predominant sugar in phloem sap collected from petioles of control leaves, sucrose (Suc) was a major sugar in the phloem sap of infected leaves. Moreover, analyses of the newly fixed (14)CO(2) revealed a high proportion of radioactive Suc in the phloem sap of infected leaves 60 min post-labeling. The alteration in phloem sap sugar composition was found in source, but not old, leaves. Moreover, elevations in Suc concentration were also evident in source leaves that did not exhibit symptoms or contain detectable amounts of virus particles. The mode by which CMV infection may cause alterations in sugar transport is discussed in terms of the mechanism by which sugars are loaded into the phloem of cucurbit plants.     

Photosynthetic electron transport in tobacco leaves infected with tobacco mosaic virus

Tobacco ( Nicotiana tabacum L. cv. Samsun) plants inoculated with different strains of tobacco mosaic virus (TMV) inducing mosaic symptoms of widely varying severity were studied with in vivo chlorophyll fluorescence. This method was used to deduce photosynthetic electron transport efficiency in relation to symptom expression. The quantum yields of photosystem II (PS II) electron transport rate were significantly diminished in virus strains inducing loss of chlorophyll. The reduction in young mosaic-diseased leaves appeared to be due in part to a reduction in the fraction of open reaction centers, whereas in older leaves exhibiting less pronounced symptoms the reduction was mainly caused by a reduced efficiency of capture of excitation energy of open PS II reaction centers. Upon infection with any of the five virus strains PS II seemed to be irreversibly damaged in the inoculated leaves and the ones directly above, indicative of a possible increased susceptibility to photoinhibition in these leaves (Somersalo and Krause 1989) even when no symptoms were apparent. Symptom expression did not appear to be related to the influence of the virus on PS II activity, because the severest effects occurred in the inoculated leaves, which either remained symptomless or developed slight yellowing only. This study demonstrates the usefulness or modulated chlorophyll fluorescence measurements for the investigation of plant-virus interactions. It is particularly important when visual symptoms are absent.     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

Expression of the tobacco mosaic virus movement protein alters starch accumulation inNicotiana benthamiana

Nicotiana benthamiana plants were transformed with the movement protein (MP) gene of tobacco mosaic virus (TMV), usingAgrobacterium-mediated transformation. Plants regenerated from the transformed cells accumulated 30-kDa MP and complemented the activity of TMV MP when infected with chimeric TMVs containing defective MR These transgenic plants displayed stunting, pale-green leaves, and starch accumulations, indicating that TMV MP altered the carbon partitioning for leaves involved in TMV cell-to-cell movement.     

Four sequence positions of the movement protein of Cucumber mosaic virus determine the virulence against cmv1‐mediated resistance in melon

The resistance to a set of strains of Cucumber mosaic virus (CMV) in the melon accession PI 161375, cultivar ‘Songwhan Charmi’, is dependent on one recessive gene, cmv1, which confers total resistance, whereas a second set of strains is able to overcome it. We tested 11 strains of CMV subgroups I and II in the melon line SC12‐1‐99, which carries the gene cmv1, and showed that this gene confers resistance to strains of subgroup II only and that restriction is not related to either viral replication or cell‐to‐cell movement. This is the first time that a resistant trait has been correlated with CMV subgroups. Using infectious clones of the CMV strains LS (subgroup II) and FNY (subgroup I), we generated rearrangements and viral chimaeras between both strains and established that the determinant of virulence against the gene cmv1 resides in the first 209 amino acids of the movement protein, as this region from FNY is sufficient to confer virulence to the LS clone in the line SC12‐1‐99. A comparison of the sequences of the strains of both subgroups in this region shows that there are five main positions shared by all strains of subgroup II, which are different from those of subgroup I. Site‐directed mutagenesis of the CMV‐LS clone to substitute these residues for those of CMV‐FNY revealed that a combination of four of these changes [the group 64–68 (SNNLL to HGRIA), and the point mutations R81C, G171T and A195I] was required for a complete gain of function of the LS MP in the resistant melon plant.     

The potential use of a viral coat protein gene as a transgene screening marker and multiple virus resistance of pepper plants coexpressing coat proteins of cucumber mosaic virus and tomato mosaic virus

Transgenic pepper plants coexpressing coat proteins (CPs) of cucumber mosaic virus (CMV-Kor) and tomato mosaic virus (ToMV) were produced by Agrobacterium-mediated transformation. To facilitate selection for positive transformants in transgenic peppers carrying an L gene, we developed a simple and effective screening procedure using hypersensitive response upon ToMV challenge inoculation. In this procedure, positive transformants could be clearly differentiated from the nontransformed plants. Transgenic pepper plants expressing the CP genes of both viruses were tested for resistance against CMV-Kor and pepper mild mottle virus (PMMV). In most transgenic plants, viral propagation was substantially retarded when compared to the nontransgenic plants. These experiments demonstrate that our transgenic pepper plants might be a useful marker system for the transgene screening and useful for classical breeding programs of developing virus resistant hot pepper plants.     

Developmental changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus

Summary Cell-to-cell communication in plants occurs through plasmodesmata, cytoplasmic channels that traverse the cell wall between neighboring cells. Plasmodesmata are also exploited by many viruses as an avenue for spread of viral progeny. In the case of tobacco mosaic virus (TMV), a virally-encoded movement protein (MP) enables the virus to move through plasmodesmata during infection. We have used thin section electron microscopy and immunocytochemistry to examine the structure of plasmodesmata in transgenic tobacco plants expressing the TMV MP. We observed a change in structure of the plasmodesmata as the leaves age, both in control and MP expressing [MP(+)] plants. In addition, the plasmodesmata of older cells of MP(+) plants accumulate a fibrous material in the central cavity. The presence of the fibers is correlated with the ability to label plasmodesmata with anti-MP antibodies. The developmental stage of leaf tissue at which this material is observed is the stage at which an increase in the size exclusion limit of the plasmodesmata can be measured in MP(+) plants. Using cell fractionation and aqueous phase partitioning studies, we identified the plasma membrane and cell wall as the compartments with which the MP stably associates. The nature of the interaction between the MP and the plasma membrane was studied using sodium carbonate and Triton X-100 washes. The MP behaves as an integral membrane protein. Identifying the mechanism by which the MP associates with plasma membrane and plasmodesmata will lead to a better understanding of how the MP alters the function of the plasmodesmata.Abbreviations MP movement protein - TMV tobacco mosaic virus     

Anti-sense RNAs of cucumber mosaic virus in transgenic plants assessed for control of the virus

Three synthetic genes for the production of anti-sense RNA to different regions of the cucumber mosaic virus (CMV) genome were constructed using virus-derived double-stranded cDNA coupled to a promoter sequence from cauliflower mosaic virus. The genes were used to transform tobacco plants by a Ti plasmid vector. Transgenic plants obtained with the three constructs produced anti-sense RNA at different levels. Plants expressing each of the three anti-sense RNAs were inoculated with CMV and their sensitivity to the virus infection was compared with the non-transformed plants. Only one plant line which expressed relatively low levels of one of the anti-sense RNAs showed resistance to CMV but other plants expressing the same or the other two antisense RNAs had similar sensitivity to CMV infection as the non-transformed plants.     

Influence of the tobacco mosaic virus 30-kDa movement protein on carbon metabolism and photosynthate partitioning in transgenic tobacco plants

Transgenic tobacco (Nicotiana tabacum L.) plants expressing the 30-kDa movement protein of tobacco mosaic virus (TMV-MP) were employed to investigate the influence of a localized change in mesophyll-bundle sheath plasmodesmal size exclusion limit on photosynthetic performance and on carbon metabolism and allocation. Under conditions of saturating irradiance, tobacco plants expressing the TMV-MP were found to have higher photosynthetic CO₂-response curves compared with vector control plants. However, this difference was significant only in the presence of elevated CO₂ levels. Photosynthetic measurements made in the green-house, under endogenous growth conditions, revealed that there was little difference between TMV-MP-expressing and control tobacco plants. However, analysis of carbon metabolites within source leaves where a TMV-MP-induced increase in plasmodesmal size exclusion limit had recently taken place established that the levels of sucrose, glucose, fructose and starch were considerably elevated above those present in equivalent control leaves. Although expression of the TMV-MP did not alter total plant biomass, it reduced carbon allocation to the lower region of the stem and roots. This difference in biomass distribution was clearly evident in the lower root-to-shoot ratios for the TMV-MP transgenic plants. Microinjection (dye-coupling) studies established that the TMV-MP-associated reduction in photosynthate delivery (allocation) to the roots was not due to a direct effect on root cortical plasmodesmata. Rather, this change appeared to result from an alteration in phloem transport from young source leaves in which the TMV-MP had yet to exert its influence over plasmodesmal size exclusion limits. These results are discussed in terms of the rate-limiting steps involved in sucrose movement into the phloem.Abbreviations PFD photon flux density - SEL size exclusion limit - TMV-MP tobacco mosaic virus movement protein This work was supported by National Science Foundation grant No. DCB-9016756 (W.J.L.) and United States-Israel Binational Science Foundation grant No. 90-00070 (S.W. and W.J.L.). Special thanks are due to Bryce Falk for the use of pathogen-free green-house space at the University of California, Davis, Plant Pathology Greenhouse Facility, and to Robert Pearcy, for the use of his gas-exchange system. R.J.H. was on sabbatical leave from the University of Rhode Island, Kingston, RI.     

Protection of transgenic tobacco plants expressing bovine pancreatic ribonuclease against tobacco mosaic virus

Transgenic tobacco plants (Nicotiana tabacum cv. SR1) expressing extracellular pancreatic ribonuclease from Bos taurus and characterized by an increased level of ribonuclease activity in leaf extracts were challenged with tobacco mosaic virus. The transgenic plants exhibited a significantly higher level of protection against the virus infection than the control non-transformed plants. The protection was evidenced by the absence (or significant delay) of the appearance of typical mosaic symptoms and the retarded accumulation of infectious virus and viral antigen. These results demonstrate that modulation of extracellular nuclease expression can be efficiently used in promoting protection against viral diseases.     

The symptom difference induced by Tobacco mosaic virus and Tomato mosaic virus in tobacco plants containing the N gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaicvirus (ToMV) are members of the genus Tobamoviruswith a world-wide distribution, and cause severe dis-eases on many economically important crops. TMVand ToMV have very close relationship and both havessRNA genome with a length of about 6400 nucleo-tides, encoding at least three nonstructural proteinsand a 17.6 kD coat protein (CP). Both 126 kD and 183kD proteins function as components of replicase, andthe 30 kD protein is involved in viral ce…     

Structural comparisons of the aggregates of tobacco mosaic virus protein

The coat protein of tobacco mosaic virus forms numerous aggregates, including the small A-protein, the disk, and two helical forms. The structures of the disk, the helical protein forms, and the virus are compared. Most of the differences are in the conformation of the chain between residues 89 and 113, which lies in the region of protein at the center of the virus, inside the RNA. It is disordered in the disk, but has a fixed conformation in the virus and the protein helices. The differences between the virus and the two helical protein forms are largely in the conformations of arginines and carboxylic acids in this region.     

Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance

Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP⁺) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP⁺ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP⁺ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.     

Inhibitory effect of fucoidan from brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus in tobacco leaves of two cultivars

The effect of fucoidan from the brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus (TMV) was investigated in the leaves of tobacco (Nicotiana tabacum L.) of two cultivars (Ksanti-nk and Samsun). In the leaves of cv. Ksanti-nk inoculated with a mixture of TMV preparation (2 μg/ml) and fucoidan (1 mg/ml), the number of local necrotic lesions induced by the virus decreased by more than 90% as compared with the leaves inoculated with the virus alone. In tobacco leaves of cv. Samsun, virulence and the concentration of the virus 3 days after inoculation with the same mixture of TMV and fucoidan were by 62 and 66%, respectively, lower than in the leaves inoculated with TMV alone. As the infection spread, the inhibitory effect of fucoidan decreased. When the leaves were treated with fucoidan before and after the inoculation with TMV, its antiviral activity was less pronounced than when a mixture of the virus and the polysaccharide was used as inoculum. Electron microscopic investigation of TMV mixed with fucoidan often showed agglutinated virions. The highest virulence of the mixture (TMV preparation, 12 μg/ml, plus fucoidan, 1 mg/ml) was observed upon its twofold dilution, and after that it decreased. It was concluded that, when the leaves were inoculated with the mixture of TMV and fucoidan, the latter affected not only the plant but the virus as well. Treatment of tobacco leaves, cv. Ksanti-nk, with actinomycin D (10 μg/ml) 24 h before the inoculation with TMV almost completely suppressed the effect of fucoidan, indicating that fucoidan acted at a gene level.     

Short-term polyamine response in TMV-inoculated hypersensitive and susceptible tobacco plants

The short-term polyamine response to inoculation, with tobacco mosaic virus (TMV), of TMV-inoculated NN (hypersensitive) and nn (susceptible) plants of Nicotiana tabacum (L.) cv. Samsun was investigated. Free and conjugated polyamine concentrations, putrescine biosynthesis, evaluated through arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities, and putrescine oxidation, via diamine oxidase (DAO) activity, were analysed during the first 24 h from inoculation. Results were compared with those of mock-inoculated control plants. In NN TMV-inoculated plants undergoing the hypersensitive response (HR), free putrescine and spermidine concentrations had increased after 5 h compared with controls; polyamine conjugates also tended to increase compared with controls. In both virus- and mock-inoculated plants, ADC and ODC activities generally increased whereas DAO activity, which was present in controls, was detectable only in traces in inoculated tissues.
In TMV-infected susceptible plants, free putrescine and spermidine concentrations were lower at 5 h relative to controls, as were polyamine conjugates. No differences were revealed in ADC and ODC activities whereas DAO activity was not detectable. These results further support the hypothesis that polyamines are involved in the response of tobacco to TMV and that, only a few hours after inoculation, the response of hypersensitive plants is distinct from that of susceptible ones.     

Plasma membrane display of anti-viral single chain Fv fragments confers resistance to tobacco mosaic virus

We tested the hypothesis that membrane-anchored anti-viral antibodies can confer viral resistance to transgenic plants. A heterologous expression system was developed for plasma membrane targeting of anti-viral antibodies using mammalian transmembrane domains. A tobacco mosaic virus (TMV) neutralizing single-chain Fv antibody fragment (scFv24) was targeted to the endoplasmic reticulum and integrated into the plasma membrane of tobacco cells, using mammalian signal peptides and membrane receptor transmembrane domains. The human platelet-derived growth factor receptor (PDGFR) transmembrane domain or the T-cell receptor -domain (TcR) transmembrane domain was fused to the C-terminus of TMV-specific scFv24 to target expression of scFv24 as an extracellularly facing plasma membrane protein. Western blot and ELISA analyses were carried out to confirm functional expression of the recombinant fusion proteins scFv24-PDGFR and scFv24-TcR in transgenic tobacco suspension cultures and transgenic plants. Immunofluorescence and electron microscopy showed that the TcR transmembrane domain targeted scFv24 to the tobacco plasma membrane. Bioassays of viral infection showed that transgenic tobacco plants expressing scFv24-TcR were resistant to TMV infection. These results demonstrated that membrane anchored anti-viral antibody fragments are functional, can be targeted to the plasma membrane in planta and are a novel approach for engineering disease-resistant crops.     

An extraction method for tobacco mosaic virus movement protein localizing in plasmodesmata

Summary. The intercellular communication by plasmodesmata (PD) is important for the growth and development of plants, and the transport of macromolecules through PD is likely to be regulated by developmental signals. While PD in the apical meristem transport macromolecules such as mRNAs, the branched PD in the mature leaf do not transport large macromolecules freely. The changes in PD during development might be important for sink-to-source changes in leaves, but the molecular mechanism is still unknown. Movement proteins (MPs) of the tobacco mosaic virus localize in the branched PD and increase the size exclusion limit, allowing transport of viral RNA. We developed a method for differential extraction of MP from isolated cell walls of transgenic tobacco leaves expressing MP or MP tagged with green-fluorescent protein. Lithium chloride at a concentration of 8 M removed filamentous structures in branched PD, the possible attachment site of MP. As some endogenous proteins were coeluted with MP by the treatment, this extraction method might be a powerful tool for investigating MP-interacting proteins in branched PD. Correspondence and reprints: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.