NTH201, a novel class II KNOTTED1-like protein, facilitates the cell-to-cell movement of Tobacco mosaic virus in tobacco

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.     

Transgenic Plants Expressing Potato Virus X ORF2 Protein (p24) Are Resistant to Tobacco Mosaic Virus and Ob Tobamoviruses

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.     

Salicylic Acid Interferes with Tobacco Mosaic Virus Replication via a Novel Salicylhydroxamic Acid-Sensitive Mechanism

Salicylic acid (SA) induces resistance to all plant pathogens, including bacteria, fungi, and viruses, but the mechanism by which SA engenders resistance to viruses is not known. Pretreatment of tobacco mosaic virus (TMV)-susceptible (nn genotype) tobacco tissue with SA reduced the levels of viral RNAs and viral coat protein accumulating after inoculation with TMV. Viral RNAs were not affected equally, suggesting that SA treatment interferes with TMV replication. Salicylhydroxamic acid (SHAM), an inhibitor of the mitochondrial alternative oxidase, antagonized both SA-induced resistance to TMV in nn genotype plants and SA-induced acquired resistance in resistant (NN genotype) tobacco. SHAM did not inhibit induction of the PR-1 pathogenesis-related protein or induction of resistance to Erwinia carotovora or Botrytis cinerea by SA. This indicates that SA induces resistance to TMV via a novel SHAM-sensitive signal transduction pathway (potentially involving alternative oxidase), which is distinct from that leading to resistance to bacteria and fungi.     

Acquired Resistance in Hypersensitive Tobacco Operates by Limiting Cell-to-Cell Spread of Virus Infection without Affecting Virus Multiplication

Localized and systemic acquired resistance against tobacco mosaic virus (TMV) or tobacco necrosis virus was induced when local lesions were produced in leaves of Xanthi-nc tobacco by mechanical inoculation with TMV. Both types of resistance were characterized by reduction in the size of lesions produced by the challenging viruses, whereas accumulation of viral antigen in lesions was slightly increased. These results, confirming previous findings relative to other hypersensitive plant virus combinations, do not support the view that an inhibitor of virus replication operates in the resistant tissues, but indicate that both types of resistance operate only against cell-to-cell spread of virus.     

Two Apoplastic alpha-Amylases Are Induced in Tobacco by Virus Infection

α-Amylase activity (EC 3.2. 1.1) is greatly increased in leaves of tobacco (Nicotiana tabacum L. cv Samsun NN) infected with tobacco mosaic virus (TMV). The kinetics of enzyme induction during the hypersensitive reaction resemble those of other hydrolases known to be pathogenesis-related proteins of tobacco. Two α-amylases were purified from TMV-infected leaves and shown to have features in common with well-characterized pathogenesis-related proteins: they are acidic monomers that can be separated upon electrophoresis on basic native gels, and they are found in the apoplastic compartment of the cell. This extra-cellular localization was demonstrated by comparing the α-amylase partition between the intercellular wash fluid and the cell extract with that of proteins of known cellular compartmentalization. These data indicate an active secretion of both α-amylases produced in tobacco upon TMV infection.     

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.     

Humans Have Antibodies against a Plant Virus: Evidence from Tobacco Mosaic Virus

Tobacco mosaic virus (TMV), a widespread plant pathogen, is found in tobacco (including cigarettes and smokeless tobacco) as well as in many other plants. Plant viruses do not replicate or cause infection in humans or other mammals. This study was done to determine whether exposure to tobacco products induces an immune response to TMV in humans. Using a sandwich ELISA assay, we detected serum anti-TMV antibodies (IgG, IgG1, IgG3, IgG4, IgA, and IgM) in all subjects enrolled in the study (20 healthy smokers, 20 smokeless-tobacco users, and 20 non-smokers). Smokers had a higher level of serum anti-TMV IgG antibodies than non-smokers, while the serum level of anti-TMV IgA from smokeless tobacco users was lower than smokers and non-smokers. Using bioinformatics, we also found that the human protein TOMM40L (an outer mitochondrial membrane 40 homolog – like translocase) contains a strong homology of six contiguous amino acids to the TMV coat protein, and TOMM40L peptide exhibited cross-reactivity with anti-TMV antibodies. People who smoke cigarettes or other tobacco products experience a lower risk of developing Parkinson’s disease, but the mechanism by which this occurs is unclear. Our results showing molecular mimicry between TMV and human TOMM40L raise the question as to whether TMV has a potential role in smokers against Parkinson’s disease development. The potential mechanisms of molecular mimicry between plant viruses and human disease should be further explored.     

杆状病毒p35基因诱导烟草产生广谱抗病机理分析

来源于昆虫病毒和动物的抗细胞凋亡基因能够诱导植物对生物或者非生物胁迫产生抗性.但其抗性机理有不同甚至相反的报道.本研究将来源于苜蓿银纹夜蛾核多角体病毒的p35基因转化烟草,T1代转化烟草Western blotting检测P35蛋白的表达,转化烟草接种烟草花叶病毒(Tobacco mosaic virus,TMV)抗病效果增强.进一步的抗病机理研究表明,转化和野生型烟草感染TMV后诱导过氧化氢积累无明显区别,野生型烟草感染24 h后出现DNA Laddering而转化烟草则没有;Western blotting结果显示PR-1蛋白表达没有显著差异.但接种另外一种病原真菌核盘茵(Sclerotiniasclerotiorum)后的RT-PCR分析结果表明,表达P35蛋白的烟草可增强感染核盘菌后PR-1基因的转录.而且表达时间提前.以上结果说明p35基因介导的广谱抗病反应的机理与接种的不同病原有关,对不同病原物的抗病机理存在差异,除抑制细胞凋亡外,还可能通过激活PR基因的表达提高对病原物的抗病能力.     

Virus Resistance Induced with Mannan Sulphates in Hypersensitive Host Plants

Mannan sulphates (MS), obtained by sulphatation of extracellular linear mannan (LM) from Rhodotorula rubra, induced resistance to tobacco mosaic virus (TMV) in hypersensitive plants, retained in isolated protoplasts. MS-induced resistance in tobacco is accompanied by a marked increase of lytic processes in cells and a decrease of total (acidic and alkaline) protein content. In addition to this, new protein components, including PR-proteins and antiviral substances, of the inhibitor of virus replication (IVR) type, appeared in treated plant tissues. MS-induced resistance resembles localized resistance induced by TMV in hypersensitive tobacco plants and could serve as an experimental model for study of the latter.     

PeaT1-induced systemic acquired resistance in tobacco follows salicylic acid-dependent pathway

Systemic acquired resistance (SAR) is an inducible defense mechanism which plays a central role in protecting plants from pathogen attack. A new elicitor, PeaT1 from Alternaria tenuissima, was expressed in Escherichia coil and characterized with systemic acquired resistance to tobacco mosaic virus (TMV). PeaT1-treated plants exhibited enhanced systemic resistance with a significant reduction in number and size of TMV lesions on wild tobacco leaves as compared with control. The quantitative analysis of TMV CP gene expression with real-time quantitative PCR showed there was reduction in TMV virus concentration after PeaT1 treatment. Similarly, peroxidase (POD) activity and lignin increased significantly after PeaT1 treatment. The real-time quantitative PCR revealed that PeaT1 also induced the systemic accumulation of pathogenesis-related gene, PR-1a and PR-1b which are the markers of systemic acquired resistance (SAR), NPR1 gene for salicylic acid (SA) signal transduction pathway and PAL gene for SA synthesis. The accumulation of SA and the failure in development of similar level of resistance as in wild type tobacco plants in PeaT1 treated nahG transgenic tobacco plants indicated that PeaT1-induced resistance depended on SA accumulation. The present work suggested that the molecular mechanism of PeaT1 inducing disease resistance in tobacco was likely through the systemic acquired resistance pathway mediated by salicylic acid and the NPR1 gene.     

The mechanism(s) of coat protein-mediated resistance against tobacco mosaic virus

The resistance of transgenic plants express genes encoding viral coat proteins to infection by the viruses from which the genes are derived was termed coat protein-mediated resistance (CP-MR) and has been demonstrated for a variety of virus/host combinations. The mechanism of CP-MR is perhaps best understood in the tobacco/TMV system. CP-MR against TMV requires accumulation of CP and does not seem to involve the induction of plant defense mechanisms. The resistance appears to be mainly based on the inhibition of virion disassembly in transgenic cells although there is evidence that a later step of infection is also affected. CP-MR of tobacco to TMV shares some features with classical cross-protection and with CP-MR in some, but not all other host/virus combinations.     

Cross protection in transgenic tobacco plants expressing a mild strain of tobacco mosaic virus

Summary Cross protection of plant viruses is a phenomenon in which plants infected with one strain of a virus are protected from the effects of superinfection by other related strains. Recently, we have succeeded in the introduction and expression of a cDNA copy of the tobacco mosaic virus (TMV) genomic RNA in transgenic tobacco plants. Using this system, we introduced a cDNA copy of a mild strain of TMV into tobacco plants. The transgenic plants did not develop any severe symptoms upon inoculation with a virulent TMV strain, indicating that these transgenic plants were cross protected against TMV infection. The system described here can be a useful model system to study the mechanism(s) of cross protection.     

Peroxidase Activity and Isoperoxidase Pattern in Tobacco Leaves Infected with Tobacco Necrosis Virus and Other Viruses Inducing Necrotic and Non-Necrotic Alterations

The level of peroxidase activity was greatly enhanced in tobacco leaves infected by tobacco necrosis virus (TNV) and other viruses which induce necrotic symptoms (TMV, ToMV and PVY^N). The intensity was related to the age of the leaves infected: absent or neglible in mature leaves and very pronounced in young growing infected leaves. On the contrary, changes in peroxidase activity were negligible when the infection was provoked by viruses which do not produce necrotic reactions (TMV and PVY^O). Analysis of the peroxidase isoenzymes, pattern in tobacco leaves infected by TNV and other necrosis-inducing viruses revealed in all cases, a slight increase in anionic (pl 3.5–3.7) and a considerable increase in moderately anionic isoenzymes particularly the pl 4.6 isoenzyme which in TNV and PVY^N-infected leaves reached levels up to 21 and 72 times the healthy control values. A considerable increase in the cationic (pl9.3–8.8) isoenzymes and the appearance of one moderately cationic isoenzyme (pl 8.2) was also detected. In leaf extracts from-virus-infected tobacco leaves with nonnecrotic response, no, or negligible alterations on the isoenzyme pattern were detected. However, infection by a fungal parasite (Erisyphe cichoracearum), which established a fully compatible, non-necrotic, interaction with tobacco leaves, like the necrosis-inducing viruses, changed the isoperoxidase pattern. The data suggest the necrotic alterations and associated changes in the peroxidase activity and isoperoxidase pattern in virus-infected leaves are not clearly related.     

Fine Structure of Healthy and TMV-Infected Tobacco Cells Grown in Tissue Culture

Three types of tobacco (Nicotiana tabacum cv. Havana 38) callus: 1) healthy stem callus, 2) TMV-infected stem callus, 3) TMV-infected leaf callus; and leaves differentiated from healthy stem callus, and from TMV-infected leaf callus were compared for fine structure. In addition, the fine structure was observed of plastids in cells of leaves differentiated from callus isolated from stem sections of TMV-infected hybrid tobacco plants (N. tabacum cv. Havana 38 ×N. glutinosa) grown under high temperature. The cytoplasmic organelles in tissue cultured cells were similar to those in cells of greenhouse-grown tobacco plants. Except for plastids, TMV infection did not noticeably affect morphologically other cellular organelles in tissue culture cells. In TMV-infected leaf callus, numerous small bodies were seen in plastid-like bodies, while vesicle-like structures were observed in the stroma of plastids in leaves differentiated from callus of hybrid tobacco inoculated with TMV. Morphological variations of mitochondria, such as swelling and vacuolization of the inner matrix, occurred frequently in TMV-infected leaf callus. Needle-like crystalline inclusions or looped inclusions composed of many fine, long filaments were considered TMV particles orientated parallel to each other. The TMV particles were detected in the cytoplasm of tissue culture cells.     

The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants

As the processing mechanism of all known potyviruses involves the activity of cysteine proteinases, we asked whether constitutive expression of a rice cysteine proteinase inhibitor gene could induce resistance against two important potyviruses, tobacco etch virus (TEV) and potato virus Y (PVY), in transgenic tobacco plants. Tobacco lines expressing the foreign gene at varying levels were examined for resistance against TEV and PVY infection. There was a clear, direct correlation between the level of oryzacystatin message, inhibition of papain (a cysteine proteinase), and resistance to TEV and PVY in all lines tested. The inhibitor was ineffective against tobacco mosaic virus (TMV) infection because processing of this virus does not involve cysteine proteinases. These results show that plant cystatins can be used against different potyviruses and potentially also against other viruses, whose replication involves cysteine proteinase activity.     

Mechanism Determining the Host Specificity of Tobacco Mosaic Virus

In order to know the mechanism controling the host specificity of tobacco mosaic virus (TMV), three species of plants showing various degrees of resistance to TMV or TMV-RNA infection were selected and the fate of infecting viral genome was studied. Extract was obtained from leaves 0.5–6 hr after inoculation of ³²P-TMV or ³²P-TMV-RNA and analyzed by sucrose density gradient centrifugation. It was found that polysomes containing infecting ³²P-RNA were formed in plants resistant to TMV to the same extent as in susceptible tobacco plants, suggesting that the host specificity of TMV is determined at a stage of viral multiplication later than the step of translation of infecting viral genome.