A Vicilin-Like Seed Storage Protein,PAP85, Is Involved in Tobacco Mosaic Virus Replication

One striking feature of viruses with RNA genomes is the modification of the host membrane structure during early infection. This process requires both virus- and host-encoded proteins; however, the host factors involved and their role in this process remain largely unknown. On infection with Tobacco mosaic virus (TMV), a positive-strand RNA virus, the filamentous and tubular endoplasmic reticulum (ER) converts to aggregations at the early stage and returns to filamentous at the late infectious stage, termed the ER transition. Also, membrane- or vesicle-packaged viral replication complexes (VRCs) are induced early during infection. We used microarray assays to screen the Arabidopsis thaliana gene(s) responding to infection with TMV in the initial infection stage and identified an Arabidopsis gene, PAP85 (annotated as a vicilin-like seed storage protein), with upregulated expression during 0.5 to 6 h of TMV infection. TMV accumulation was reduced in pap85-RNA interference (RNAi) Arabidopsis and restored to wild-type levels when PAP85 was overexpressed in pap85-RNAi Arabidopsis. We did not observe the ER transition in TMV-infected PAP85-knockdown Arabidopsis protoplasts. In addition, TMV accumulation was reduced in PAP85-knockdown protoplasts. VRC accumulation was reduced, but not significantly (P = 0.06), in PAP85-knockdown protoplasts. Coexpression of PAP85 and the TMV main replicase (P126), but not their expression alone in Arabidopsis protoplasts, could induce ER aggregations.     

Virus-induced virus inhibitors from systemically virus-infected plants

After infection ofNicotiana tabacum cv. Samsun with tobacco mosaic virus (TMV) crude extracts from dark-green spots of upper leaves had a more strongly marked inhibitory effect upon TMV addedin vitro than crude extracts from the surrounding light-green tissue. Likewise, crude extracts from leaves ofNicotiana tabacum cv. Samsun showing recovery after infection with tobacco ringspot virus (TRV) were seen to have a marked inhibitory effect on TMV addedin vitro. The results obtained suggest that virus inhibitors are produced after virus infections not only in hypersensitive hosts but also in systemic hosts. Necrotizing processes are not an indispensable prerequisite of the production of virus-induced virus inhibitors.     

Interaction of cucumber mosaic virus and potato virus y with tobacco mosaic virus

A study was performed on the interaction of cucumber mosaic virus (CMV) of potato virus Y (PVY) with tobacco mosaic virus (TMV). Interference was evaluated using tobacco plantsNicotiana tabacum cv. Java responding to CMV and PVY with a systemic infection and to TMV with local necrotic lesions. The decrease in TMV — induced lesion number gave evidence of a decrease in susceptibility caused by the previous infection with CMV or PVY, the decrease of lesion enlargement demonstrated a decreased TMV reproduction in the plants previously infected with CMV or PVY. The interference concerned was incomplete, as evaluated from reproduction of the challenging TMV and from the decrease in susceptibility of the host to TMV brought about by the first infection with CMV or PVY.     

Pathogen-inducible CaUGT1 is involved in resistance response against TMV infection by controlling salicylic acid accumulation

Capsicum annuum L. Bugang exhibits a hypersensitive response against Tobacco mosaic virus (TMV) P₀ infection. The C. annuumUDP-glucosyltransferase 1 (CaUGT1) gene was upregulated during resistance response to TMV and by salicylic acid, ethephon, methyl viologen, and sodium nitroprusside treatment. When the gene was downregulated by virus-induced gene silencing, a delayed HR was observed. In addition, free and total SA concentrations in the CaUGT1-downregulated hot pepper were decreased by 52% and 48% compared to that of the control plants, respectively. This suggested that the CaUGT1 gene was involved in resistance response against TMV infection by controlling the accumulation of SA.     

灰树花中一种抗烟草花叶病毒的蛋白质的纯化及其性质

通过硫酸铵分级沉淀、阴离子交换柱层析及凝胶过滤柱层析,从灰树花子实体中分离到了一种具有抑制烟草花叶病毒（TMV）侵染活性的热稳定蛋白——GFAP.经常规凝胶电泳和等电聚焦电泳显示为单一条带,而SDS-聚丙烯酰胺凝胶电泳结果证明该蛋白质含有两个亚基,其分子质量分别为34 ku、40 ku.等电聚焦测定蛋白质pI为3.76,含糖量约为2%.GFAP的40 ku条带N端氨基酸序列为ACCVPSVTEFENAINSDPVM,将其与GenBank中的氨基酸序列检索比较后没有发现同源序列,预示可能为一新的氨基酸序列.GFAP与TMV混合接种心叶烟,当GFAP浓度为32 mg/L时即可完全抑制浓度为10 mg/L的TMV的侵染,而4 mg/L的GFAP对浓度为40 mg/L的TMV的抑制率仍可达60%以上.     

Effect of chitosan on tobacco mosaic virus（TMV） accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus（TMV） in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV（2 μg/mL） and chitosan（1 mg/mL） were lower in the early period of infection（3 days after inoculation）, by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases（proteases, RNases） in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid（PTA）-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles（18 nm in diameter, 300 nm long）, these suspensions contained abnormal（swollen, “thin” and “short”） virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.     

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.     

Induction of Systemic Resistance to Tobacco Powdery Mildew by Tobacco Mosaic Virus, Tobacco Necrosis Virus or Ethephon

Local infections of either TMV or TNV in tobacco plants cv. Havana 425 (hypersensitive to TMV) proved effective in inducing systemic resistance to subsequent inoculation with the powdery mildew fungus Erysiphe cichoracearum DC. The proportion of leaf surface invaded by this pathogen and the amount of conidia it produced were both significantly lower in virus inoculated plants than in non-inoculated controls. However, the decrease in sporulation rate was less regularly observed than the reduction in leaf area infected. TMV was more effective than TNV in protecting tobacco plants from powdery mildew. E. cichoracearum is thus added to the list of challenge pathogens to which TMV or TNV are known to induce resistance in the host plants. Necrotic lesions caused to the leaves by local treatment with Ethephon (an ethylene-releasing compound) also conferred to tobacco some degree of systemic resistance to the same fungal pathogen, more frequently visible as a reduction of leaf area invaded. The protection due to the Ethephon lesions was in present experiments less marked than that of TMV. No effects against subsequent powdery mildew infection were obtained when point freeze necrotic lesions were provoked on the plants.     

Protein composition of tobacco leaves with antiviral resistance induced by activators of defense responses and TMV infection

In tobacco (Nicotiana tabacum L.) plants of hypersensitive cv. Samsun NN, a capability of necrosis lesion formation and protein patterns were studied after induction of antiviral resistance by defense responses activators (DRA) (arachidonic acid, ubiquinone 50, and vitamin E) and by infection with tobacco mosaic virus (TMV). DRA and TMV improved both local and systemic leaf resistance to TMV. Native protein electrophoresis demonstrated differences in the composition of leaf proteins extracted under acidic and alkaline conditions. SDS-PAGE revealed proteins accumulated during the development of systemic antiviral resistance after lower leaf treatments with DRA and of local resistance induced by pretreatment with TMV. It was shown that various DRA affected protein patterns similarly, whereas TMV infection resulted in other changes. It is supposed that different pathways function in tobacco plants during induction of systemic resistance by DRA and TMV infection.     

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.     

Effect of tobacco mosaic virus infection of levels of soluble superoxide dismutase (SOD) in nicotiana tabacum and nicotiana glutinosa leaves

The activity of superoxide dismutase (SOD: E.C. 1.15.1.1) was evaluated on Nicotiana tabacum and Nicotiana glutinosa leaf tissue after Tobacco Mosaic Virus (TMV) infection. Significant increase in extracted SOD appeared to be directly related to the appearance of necrotic and systemic symptoms in hypersensitive (N. glutinosa and N. tabacum cv. Havana 425) and susceptible (N. tabacum cv. Bright BC 60) leaves, respectively. SOD activity did not change significantly during the replication of TMV in the inoculated susceptible leaves up to 4 days after inoculation. Both cyanide-insensitive (2 days after inoculation) and sensitive (3–4 days after inoculation) enzymes increased during the expression of the hypersensitivity. Only cyanide-sensitive enzyme increased in systematically infected leaves. SOD and peroxidase increased simultaneously and the enhancement of peroxidase was higher than that of SOD. The values of peroxidase greatly exceeded that of SOD only in the hypersensitive leaves during local lesion differentiation. In N. tabacum leaves 4 clear SOD bands were separated by polyacrylamide gel electrophoresis: 3 cyanide-sensitive (Cu,Zn enzyme) and 1 cyanide-insensitive, while N. glutinosa had 3 bands: 2 cyanide-sensitive and 1 cyanide-insensitive. The cyanide-insensitive band, both in N. tabacum and N. glutinosa, was sensitive to H₂O₂ and insensitive to chloroform-ethanol treatment and thus supposed to be Fe enzyme. The infection did not induce change in the electrophoretic pattern of SOD enzymes.In summary, our results indicate that the pathogenic alteration caused by TMV infection both in the compatible and in the incompatible combinations are characterized by an induction of SOD activity, particularly cyanide-sensitive Cu,Zn-SOD. The connection between the induction of SOD and a possible activation of O₂⁻ production in the hypersensitive tissue following TMV infection is discussed.     

Experimental Virus Evolution Reveals a Role of Plant Microtubule Dynamics and TORTIFOLIA1/SPIRAL2 in RNA Trafficking

The cytoskeleton is a dynamic network composed of filamentous polymers and regulatory proteins that provide a flexible structural scaffold to the cell and plays a fundamental role in developmental processes. Mutations that alter the spatial orientation of the cortical microtubule (MT) array of plants are known to cause important changes in the pattern of cell wall synthesis and developmental phenotypes; however, the consequences of such alterations on other MT-network-associated functions in the cytoplasm are not known. In vivo observations suggested a role of cortical MTs in the formation and movement of Tobacco mosaic virus (TMV) RNA complexes along the endoplasmic reticulum (ER). Thus, to probe the significance of dynamic MT behavior in the coordination of MT-network-associated functions related to TMV infection and, thus, in the formation and transport of RNA complexes in the cytoplasm, we performed an evolution experiment with TMV in Arabidopsis thaliana tor1/spr2 and tor2 mutants with specific defects in MT dynamics and asked whether TMV is sensitive to these changes. We show that the altered cytoskeleton induced genetic changes in TMV that were correlated with efficient spread of infection in the mutant hosts. These observations demonstrate a role of dynamic MT rearrangements and of the MT-associated protein TORTIFOLIA1/SPIRAL2 in cellular functions related to virus spread and indicate that MT dynamics and MT-associated proteins represent constraints for virus evolution and adaptation. The results highlight the importance of the dynamic plasticity of the MT network in directing cytoplasmic functions in macromolecular assembly and trafficking and illustrate the value of experimental virus evolution for addressing the cellular functions of dynamic, long-range order systems in multicellular organisms.     

Type I J-Domain NbMIP1 Proteins Are Required for Both Tobacco Mosaic Virus Infection and Plant Innate Immunity

Tm-2² is a coiled coil-nucleotide binding-leucine rich repeat resistance protein that confers durable extreme resistance against Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) by recognizing the viral movement protein (MP). Here we report that the Nicotiana benthamiana J-domain MIP1 proteins (NbMIP1s) associate with tobamovirus MP, Tm-2² and SGT1. Silencing of NbMIP1s reduced TMV movement and compromised Tm-2²-mediated resistance against TMV and ToMV. Furthermore, silencing of NbMIP1s reduced the steady-state protein levels of ToMV MP and Tm-2². Moreover, NbMIP1s are required for plant resistance induced by other R genes and the nonhost pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In addition, we found that SGT1 associates with Tm-2² and is required for Tm-2²-mediated resistance against TMV. These results suggest that NbMIP1s function as co-chaperones during virus infection and plant immunity.     

Hormonal status of tobacco variety Samsun NN exposed to synthetic coenzyme Q10 (ubiquinone 50) and TMV infection

Hormonal system status has been analyzed in leaf disks of hypersensitive tobacco Nicotiana tabacum L. variety Samsun NN during the development of resistance to tobacco mosaic virus (TMV) induced by synthetic coenzyme Q₁₀ (ubiquinone 50). The absolute and relative content of abscisic acid (ABA), indoleacetic acid (IAA), and cytokinins (CKs) was determined after the exposure of leaves to Q₁₀ solution and the subsequent TMV infection. In plants not treated with Q₁₀, CK content increased about 2.5 times 1 day after TMV infection, while a significant increase in the ABA level and a decrease in the IAA level were observed only after 2 days. In the dynamics, Q₁₀ treatment had a protective antiviral effect, significantly decreased the ABA level, and increased the IAA level in sensitized plants compared to nonsensitized ones.     

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

Background

Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use.

Results

We have constructed a Cauliflower mosaic virus (CaMV) 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration) of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV) resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI). The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date.

Conclusion

These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.     

Reproduction of sugar beet mosaic and tobacco mosaic viruses in anthocyanized beet plants

During our studies on the interaction of anthocyanins and plant virus diseases, reproduction of sugar beet mosaic (SBMV) and tobacco mosaic viruses (TMV) was investigated. Experiments were carried out in leaves of sugar beet,Beta vulgaris cv. Dobrovicka N and its spontaneous anthocyanized mutant. SBMV induces a systemic infection while TMV is responsible for primary local symptoms in sugar beet leaves only. Our quantitative analyses onAmaranthus caudatus L. andChenopodium quinoa Wilid. showed a significant decrease in concentration of SBMV in juice extracted from anthocyanized beet plants as compared with extracts from normal green infected plants. Significant differences were also obtained when SBMV — containing juice was tested in mixtures with healthy extracts from anthocyanized and normal green plants. Also the intensity of TMV symptoms in beet leaves was considerably decreased in leaves of antho-eyanized plants.     

Silicon delays Tobacco ringspot virus systemic symptoms in Nicotiana tabacum

Soluble silicon (Si) provides protection to plants against a variety of abiotic and biotic stress. However, the effects of Si on viral infections are largely unknown. To investigate the role of Si in viral infections, hydroponic studies were conducted in Nicotiana tabacum with two pathogens: Tobacco ringspot virus (TRSV) and Tobacco mosaic virus (TMV). Plants grown in elevated Si showed a delay in TRSV systemic symptom formation and a reduction in symptomatic leaf area, compared to the non-supplemented controls. TRSV-infected plants showed significantly higher levels of foliar Si compared to mock-inoculated plants. However, the Si effect appeared to be virus-specific, since the element did not alter TMV symptoms nor did infection by this virus alter foliar Si levels. Hence, increased foliar Si levels appear to correlate with Si-modulated protection against viral infection. This is all the more intriguing since N. tabacum is classified as a low Si accumulator.     

Reduced Photosystem II Activity and Accumulation of Viral Coat Protein in Chloroplasts of Leaves Infected with Tobacco Mosaic Virus

We previously reported (A Reinero, RN Beachy 1986 Plant Mol Biol 6:291-301) that coat protein (CP) of tobacco mosaic virus (TMV) accumulates in chloroplasts of systemically infected leaves. To determine the significance of such interaction we examined electron transport rates in chloroplasts containing different levels of TMV-CP. Tobacco (Nicotiana tabacum L.) plants were infected with either a TMV strain inducing chlorosis or with a strain inducing mild symptoms, and both the accumulation pattern of TMV-CP inside chloroplasts as well as the rates of photosynthetic electron transport were followed. The CP of the TMV strain inducing chlorosis was detected inside chloroplasts 3 days after infection, and thereafter accumulated at a rapid rate, first in the stroma and then in the thylakoid membranes. On the other hand, the CP of the TMV strain that caused only mild symptoms accumulated in chloroplasts to lower levels and little CP was associated with the thylakoids. In vivo and in vitro measurements of electron transport revealed that photosystem II activity was inhibited in plants infected with the aggressive TMV strain while no reduction was observed in plants infected with the mild strain. The capacity of chloroplasts to synthesize proteins was equivalent in organelles isolated from healthy and virus-infected leaves. The possibility that a large accumulation of TMV-CP inside chloroplasts may affect photosynthesis in virus-infected plants by inhibiting photosystem II activity is discussed.     

Pathogenesis-related functions of plant β-1,3-glucanases investigated by antisense transformation — a review

Plant β-1,3-glucanases (βGlu) have been implicated in several physiological and developmental processes, e.g., cell division, microsporogenesis, pollen germination, fertilization and seed germination. These enzymes, particularly the antifungal class-I vacuolar isoforms, are also believed to be part of the defences of plants against fungal infection. The function of βGlu in tobacco and Nicotiana sylvestris has been investigated by antisense transformation. Transformation with GLA, the gene encoding the A isoform of tobacco class-I βGlu, in reverse orientation regulated by the strong cauliflower mosaic virus 35S RNA promoter effectively and specifically blocked the induction of class-I βGlu. This induction was in response to ethylene treatment and following infection with the pathogenic fungus, Cercospora nicotianae, tobacco mosaic virus (TMV) and tobacco necrosis virus (TNV). Nevertheless, the plants compensated for this deficiency by producing a functionally equivalent (i.e., ‘ersatz’) enzyme or enzymes. The fact that compensation occurred specifically in response to infection suggests that βGlu activity has an important role in pathogenesis. Antisense transformation substantially reduced lesion size and number in virus-infected local-lesion hosts. These results suggest novel antisense-based strategies for protecting plants against virus infection. They also raise the intriguing possibility that viruses use a defence mechanism of the host, production of antifungal βGlu, to promote their own replication and spread.     

Turnip vein clearing virus movement protein nuclear activity: Do Tobamovirus movement proteins play a role in immune response suppression?

Plant viruses'' cell-to-cell movement requires the function of virally encoded movement proteins (MPs). The Tobamovirus, Tobacco mosaic virus (TMV) has served as the model virus to study the activities of single MPs. However, since TMV does not infect the model plant Arabidopsis thaliana I have used a related Tobamovirus, Turnip vein-clearing virus (TVCV). I recently showed that, despite belonging to the same genus, the behavior of the 2 viruses MPs differ significantly during infection. Most notably, MP^TVCV, but not MP^TMV, targets the nucleus and induces the formation of F actin-containing filaments that associate with chromatin. Mutational analyses showed that nuclear localization of MP^TVCV was necessary for TVCV local and systemic infection in both Nicotiana benthamiana and Arabidopsis. In this addendum, I propose possible targets for the MP^TVCV nuclear activity, and suggest viewing MPs as viral effector-like proteins, playing a role in the inhibition of plant defense.