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…     

Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.     

Interactions between tobacco mosaic virus and the tobacco N gene

The interaction between tobacco mosaic virus (TMV) and tobacco harbouring the N gene is a classical system for studying gene-for-gene interactions in disease resistance. The N gene confers resistance to TMV by mediating defence responses that function to limit viral replication and movement. We isolated the N gene and determined that N belongs to the nucleotide-binding-site-leucine-rich-repeat (NBS-LRR) class of plant disease resistance genes, and encodes both full-length and truncated proteins. Sequence homologies and mutagenesis studies indicated a signalling role for the N protein similar to that seen for proteins involved in defence responses in insects and mammals. The N gene confers resistance to TMV in transgenic tomato, demonstrating the use of the NBS-LRR class of disease resistance genes in engineering crop resistance. From the pathogen side of this interaction, the TMV 126 kDa replicase protein has been implicated as the avirulence factor that triggers N-mediated defence responses. We employed Agrobacterium-mediated expression strategies to demonstrate that expression of the putative helicase region of the replicase protein is sufficient to elicit N-mediated defences. The thermosensitivity of the N-mediated response to TMV is retained when induced by expression of this replicase fragment. Thus, both components of this gene-for-gene interaction are now available for studies that address the molecular mechanisms involved in N-mediated TMV resistance.     

Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus

The tobacco N gene confers resistance to tobacco mosaic virus (TMV) and encodes a Toll-interleukin-1 receptor/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class protein. We have developed and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system to investigate the role of tobacco candidate genes in the N-mediated signalling pathway. To accomplish this we generated transgenic Nicotiana benthamiana containing the tobacco N gene. The transgenic lines exhibit hypersensitive response (HR) to TMV and restrict virus spread to the inoculated site. This demonstrates that the tobacco N gene can confer resistance to TMV in heterologous N. benthamiana. We have used this line to study the role of tobacco Rar1-, EDS1-, and NPR1/NIM1- like genes in N-mediated resistance to TMV using a TRV based VIGS approach. Our VIGS analysis suggests that these genes are required for N function. EDS1-like gene requirement for the N function suggests that EDS1 could be a common component of bacterial, fungal and viral resistance signalling mediated by the TIR-NBS-LRR class of resistance proteins. Requirement of Rar1- like gene for N-mediated resistance to TMV and some powdery mildew resistance genes in barley provide the first example of converging points in the disease resistance signalling pathways mediated by TIR-NBS-LRR and CC-NBS-LRR proteins. The TRV based VIGS approach as described here to study N-mediated resistance signalling will be useful for the analysis of not only disease resistance signalling pathways but also of other signalling pathways in genetically intractable plant systems.     

Molecular cloning of a metallothionein-like gene from Nicotiana glutinosa L. and its induction by wounding and tobacco mosaic virus infection.

The cloning and characterization of genes expressed in plant disease resistance could be an initial step toward understanding the molecular mechanisms of disease resistance. A metallothionein-like gene that is inducible by tobacco mosaic virus and by wounding was cloned in the process of subtractive cloning of disease resistance-response genes in Nicotiana glutinosa. One 530-bp cDNA clone (KC9-10) containing an open reading frame of 81 amino acids was characterized. Genomic Southern blot hybridization with the cDNA probe revealed that tobacco metallothionein-like genes are present in few or in one copy per diploid genome. Northern blot hybridization detected strong induction of a 0.5-kb mRNA by wounding and tobacco mosaic virus infection, but only mild induction was detected when copper was tested as an inducer. Methyl jasmonate, salicylic acid, and ethylene were also tested as possible inducers of this gene, but they had no effect on its expression. The possible role of this gene in wounded and pathogen-stressed plants is discussed.     

Milestones in the research on tobacco mosaic virus

Beijerinck's (1898) recognition that the cause of tobacco mosaic disease was a novel kind of pathogen became the breakthrough which eventually led to the establishment of virology as a science. Research on this agent, tobacco mosaic virus (TMV), has continued to be at the forefront of virology for the past century. After an initial phase, in which numerous biological properties of TMV were discovered, its particles were the first shown to consist of RNA and protein, and X-ray diffraction analysis of their structure was the first of a helical nucleoprotein. In the molecular biological phase of research, TMV RNA was the first plant virus genome to be sequenced completely, its genes were found to be expressed by cotranslational particle disassembly and the use of subgenomic mRNA, and the mechanism of assembly of progeny particles from their separate parts was discovered. Molecular genetical and cell biological techniques were then used to clarify the roles and modes of action of the TMV non-structural proteins: the 126 kDa and 183 kDa replicase components and the 30 kDa cell-to-cell movement protein. Three different TMV genes were found to act as avirulence genes, eliciting hypersensitive responses controlled by specific, but different, plant genes. One of these (the N gene) was the first plant gene controlling virus resistance to be isolated and sequenced. In the biotechnological sphere, TMV has found several applications: as the first source of transgene sequences conferring virus resistance, in vaccines consisting of TMV particles genetically engineered to carry foreign epitopes, and in systems for expressing foreign genes. TMV owes much of its popularity as a research mode to the great stability and high yield of its particles. Although modern methods have much decreased the need for such properties, and TMV may have a less dominant role in the future, it continues to occupy a prominent position in both fundamental and applied research.     

The suppressor of transgene RNA silencing encoded by Cucumber mosaic virus interferes with salicylic acid-mediated virus resistance

The Cucumber mosaic virus (CMV)-encoded 2b protein (Cmv2b) is a nuclear protein that suppresses transgene RNA silencing in Nicotiana benthamiana. Cmv2b is an important virulence determinant but nonessential for systemic spread in N. glutinosa, in contrast to its indispensable role for systemic infections in cucumber. Here, we report that Cmv2b became essential for systemic infections in older N. glutinosa plants or in young seedlings pretreated with salicylic acid (SA). Expression of Cmv2b from the genome of either CMV or Tobacco mosaic virus significantly reduced the inhibitory effect of SA on virus accumulation in inoculated leaves and systemic leaves. A close correlation is demonstrated between Cmv2b expression and a reduced SA-dependent induction of the alternative oxidase gene, a component of the recently proposed SA-regulated antiviral defense. These results collectively reveal a novel activity of Cmv2b in the inhibition of SA-mediated virus resistance. We used a N. tabacum line expressing a bacterial nahG transgene that degrades SA to provide evidence for a Cmv2b-sensitive antiviral defense mechanism in tobacco in which SA acts as a positive modifier but not as an essential component. We propose that SA induces virus resistance by potentiating a RNA-silencing antiviral defense that is targeted by Cmv2b.     

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.     

Cucumber mosaic virus coat protein-mediated protection

Transgenic tobacco plants (CP +) that express the coat protein gene of cucumber mosaic virus (CMV)-Y strain were highly protected from infection with either CMV virions or CMV RNA, while transgenic protoplasts were also protected from infection with CMV virions but not with CMV RNA. CP + plants showed greater susceptibility to infection with satellite RNA-free CMV-Y than CMV-Y containing satellite RNA. At temperatures above 30°C, CP + plants did not or poorly resist infection with CMV. Elevated temperature affected the accumulation of CP rather than its mRNA, suggesting that CP molecules are mainly involved in virus resistance in CP + plants.     

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.     

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.     

Nicotiana benthamiana asparagine synthetase associates with IP-L and confers resistance against tobacco mosaic virus via the asparagine-induced salicylic acid signalling pathway

Asparagine synthetase is a key enzyme that catalyses the conversion of amide groups from glutamine or ammonium to aspartate, which leads to the generation of asparagine. However, the role of asparagine synthetase in plant immunity remains largely unknown. Here, we identified a Nicotiana benthamiana asparagine synthetase B (NbAS-B) that associates with tomato mosaic virus coat protein-interacting protein L (IP-L) using the yeast two-hybrid assay and examined its role in tobacco mosaic virus (TMV) resistance. The association of IP-L with NbAS-B was further confirmed by in vivo co-immunoprecipitation, luciferase complementation imaging, and bimolecular fluorescence complementation assays. IP-L and NbAS-B interact in the nucleus and cytosol and IP-L apparently stabilizes NbAS-B, thus enhancing its accumulation. The expressions of IP-L and NbAS-B are continuously induced on TMV-green fluorescent protein (GFP) infection. Co-silencing of IP-L and NbAS-B facilitates TMV-GFP infection. Overexpression of NbAS-B in tobacco reduces TMV-GFP infection by significantly improving the synthesis of asparagine. Furthermore, the external application of asparagine significantly inhibits the infection of TMV-GFP by activating the salicylic acid signalling pathway. These findings hold the potential for the future application of asparagine in the control of TMV.     

Polyacrylic acid-induced resistance to tobacco mosaic virus in tobacco cv. Xanthi

Polyacrylic acid (PA) of molecular weight 1700A, 1700B and 3500 caused resistance to infection with tobacco mosaic virus in tobacco cv. Xanthi-nc, when sprayed on the leaves or watered into the soil. The numbers of lesions produced in the treated plants were between 27 and 97% fewer than in the untreated plants depending on the concentration of PA, its molecular weight and the method of application. Some resistance was caused against potato virus X and potato virus Y but only at concentrations that were harmful to the plants. It appears that PA activates a mechanism responsible for localizing viruses in hypersensitive plants.