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Byline: T. E. Koshkina (1), E. N. Baranova (1), E. A. Usacheva (1), S. K. Zavriev (1) Keywords: tobacco mosaic virus; coat protein; kinetics of long-distance virus transport Abstract: A mutation resulting in substitution of positively charged Lys53 with negatively charged Glu in the coat protein was introduced in the infectious cDNA copy of the genome of wild-type tobacco mosaic virus strain U1. Kinetic analysis of long-distance virus transport in plants showed that systemic spread of the mutant virus was delayed by 5--6 days as compared with the wild-type one. On evidence of RNA sequencing in the mutant progeny, Glu50 of the coat protein was substituted with Lys after passage I to compensate for the loss of the positive charge at position 53. Electron microscopy revealed atypical inclusions (rodlike structures, multiple electron-dense globular particles) in the nuclear interchromatin space of leaf mesophyll cells infected with the mutant but not with the wild-type virus. Author Affiliation: (1) Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, Moscow, 127550, Russia Article History: Registration Date: 12/10/2004

Byline: E. V. Lukashina (1), G. A. Badun (1), V. M. Fedoseev (1), N. V. Fedorova (2), A. L. Ksenofontov (2), L. A. Baratova (2), E. N. Dobrov (2) Keywords: coat protein; tobacco mosaic virus; tritium planigraphy; necrotic response Abstract: Mutant ts21-66 of the tobacco mosaic virus (TMV) differs from the wild-type TMV-U1 by two mutations (Ile21 [right arrow] Thr and Asp66 [right arrow] Gly) in the coat protein (CP) gene and in symptoms produced in infected N' plants. The CP structure in TMV-U1 and ts21-66 virions was probed by tritium planigraphy. Compared with the wild-type CP, labeling of the N-terminal region of mutant CP was half as high and suggested its greater shielding. The role of this CP region in virus interactions with the N' resistance system is discussed. Author Affiliation: (1) Department of Chemistry, Moscow State University, Moscow, 119899, Russia (2) Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119899, Russia Article History: Registration Date: 08/10/2004

Salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two distinct members of the mitogen-activated protein (MAP) kinase family, are activated in tobacco resisting infection by tobacco mosaic virus (TMV). WIPK activation by TMV depends on the disease-resistance gene N because infection of susceptible tobacco not carrying the N gene failed to activate WIPK. Activation of WIPK required not only posttranslational phosphorylation but also a preceding rise in its mRNA and de novo synthesis of WIPK protein. The induction by TMV of WIPK mRNA and protein also occurred systemically. Its activation at the mRNA, protein, and enzyme levels was independent of salicylic acid. The regulation of WIPK at multiple levels by an N gene-mediated signal(s) suggests that this MAP kinase may be an important component upstream of salicylic acid in the signal-transduction pathway(s) leading to local and systemic resistance to TMV.

Salicylic acid (SA) plays an important role in signaling the activation of plant defense responses against pathogen attack including induction of pathogenesis-related (PR) proteins. To gain further insight into the SA-mediated signal transduction pathway, we have isolated and characterized a tobacco mosaic virus (TMV)-inducible myb oncogene homolog (myb1) from tobacco. The myb1 gene was induced upon TMV infection during both the hypersensitive response and development of systemic acquired resistance in the resistant tobacco cultivar following the rise of endogenous SA, but was not activated in the susceptible cultivar that fails to accumulate SA. The myb1 gene was also induced by incompatible bacterial pathogen Pseudomonas syringae pv. syringae during the hypersensitive response. Exogenous SA treatment rapidly (within 15 min) activated the expression of myb1 in both resistant and susceptible tobacco cultivars with the subsequent induction of PR genes occurring several hours later. Biologically active analogs of SA and 2,6-dichloroisonicotinic acid (a synthetic functional analog of SA), which induce PR genes and enhanced resistance, also activated the myb1 gene. In contrast, biologically inactive analogs were poor inducers of myb1 gene expression. Furthermore, the recombinant Myb1 protein was shown to specifically bind to a Myb-binding consensus sequence found in the promoter of the PR-1a gene. Taken together, these results suggest that the tobacco myb1 gene encodes a signaling component downstream of SA that may participate in transcriptional activation of PR genes and plant disease resistance.

To fully understand vascular transport of plant viruses, the viral and host proteins, their structures and functions, and the specific vascular cells in which these factors function must be determined. We report here on the ability of various cDNA-derived coat protein (CP) mutants of tobacco mosaic virus (TMV) to invade vascular cells in minor veins of Nicotiana tabacum L. cv. Xanthi nn. The mutant viruses we studied, TMV CP-O, U1mCP15-17, and SNC015, respectively, encode a CP from a different tobamovirus (i.e., from odontoglossum ringspot virus) resulting in the formation of non-native capsids, a mutant CP that accumulates in aggregates but does not encapsidate the viral RNA, or no CP. TMV CP-O is impaired in phloem-dependent movement, whereas U1mCP15-17 and SNC015 do not accumulate by phloem-dependent movement. In developmentally-defined studies using immunocytochemical analyses we determined that all of these mutants invaded vascular parenchyma cells within minor veins in inoculated leaves. In addition, we determined that the CPs of TMV CP-O and U1mCP15-17 were present in companion (C) cells of minor veins in inoculated leaves, although more rarely than CP of wild-type virus. These results indicate that the movement of TMV into minor veins does not require the CP, and an encapsidation-competent CP is not required for, but may increase the efficiency of, movement into the conducting complex of the phloem (i.e., the C cell/sieve element complex). Also, a host factor(s) functions at or beyond the C cell/sieve element interface with other cells to allow efficient phloem-dependent accumulation of TMV CP-O.

It has been proposed that cloned plant disease resistance genes could be transferred from resistant to susceptible plant species to control important crop plant diseases. The recently cloned N gene of tobacco confers resistance to the viral pathogen, tobacco mosaic virus. We generated transgenic tomato plants bearing the N gene and demonstrate that N confers a hypersensitive response and effectively localizes tobacco mosaic virus to sites of inoculation in transgenic tomato, as it does in tobacco. The ability to reconstruct the N-mediated resistance response to tobacco mosaic virus in tomato demonstrates the utility of using isolated resistance genes to protect crop plants from diseases, and it demonstrates that all the components necessary for N-mediated resistance are conserved in tomato.

The resistance gene N of tobacco, which mediates the resistance to the tobacco mosaic virus (TMV), is isolated by transposon tagging. The tagging uses the maize activator transposon, which confers the genomic DNA fragment with an N gene to TMV susceptible tobacco. The plant disease resistance genes identify and combat the invading pathogens by triggering host defense responses. This is performed by the resistance gene N, which encodes the 131.4 kDa protein with an amino acid terminal domain.

The genes of the tobacco mosaic virus (TMV) which are expressed via subgenomic mRNAs are regulated independently. Among these genes are one coding for a 30-kDa cell-to-cell movement protein and others coding for coat protein. Manipulations of the genomic postions of these genes showed that placement of these genes closer to the viral 3' terminus resulted in higher gene expression.Therefore, position effect plays a major role in the regulation of TMV gene expression.

The single-molecule force spectroscopy technique is employed to explain the RNA-coat protein interactions taking place in the intact tobacco mosaic virus. The various effects of pulling speed, as well as pH on these interactions are also analyzed.

The efficiency of energy transfer within systems containing a high ratio of donors to acceptors has shown a strong dependence on the tobacco mosaic virus coat protein (TMVP) assembly state along with the availability of redundant energy transfer pathways that can circumvent defect sites. The simulations have shown that important differences in excited state dissipation rates have contributed to the greater efficiency of the rods and that statistical variation in the assembly process play a more minor role.

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.

HSP101, a tobacco mosaic virus (TMV) 102-kD protein homologous to the HSP101/HSP104/ClpB heat shock protein family, acts as a specific translation-regulation protein. Its activity is controlled by nutrient status. RNA-binding and translation regulatory activites of HSP101 are not active in respiring cells or in those subject to limits on nutrients, but thermotolerance functions of the protein are unaffected in those circumstances. A protein necessary for specific translation enhancement of capped mRNAs has never been identified before nor has a translation-regulation function for a heat shock protein been reported. No functional distinction between the two eIF4G proteins found in eukaryotes has been made before. HSP101 binds the 68-nucleotide 5' leader which is partly responsible for efficient translation of TMV mRNA. The leader has no guanosine residues and has a central poly(CAA) region needed for stimulation of translation.