To Tobacco Mosaic Virus Research Articles

Transglutaminase activity changes during the hypersensitive reaction, a typical defense response of tobacco NN plants to TMV

The occurrence of glutamyl polyamines (PAs) and changes in activity and levels of transglutaminase (TGase, EC 2.3.2.13), the enzyme responsible for their synthesis, are reported during the progression of the hypersensitive reaction (HR) of resistant NN tobacco plants (Nicotiana tabacum L. cv. Samsun) to tobacco mosaic virus (TMV). Mature leaves of tobacco were collected over 0-72 h after inoculation with TMV or phosphate buffer (mock). In vivo synthesis of polyamine glutamyl derivatives (glutamyl PAs), catalyzed by TGase activity, was evaluated after supplying labeled putrescine (Pu, a physiological substrate of TGase) to leaves. Results show that, starting from 24 h, mono-(gamma-glutamyl)-Pu and bis-(gamma-glutamyl)-Sd were recovered in TMV-inoculated samples but not in mock-inoculated ones; 2 days later, in the former, the amount of glutamyl derivatives further increased. An in vitro radiometric assay showed that, in TMV-inoculated leaves, TGase activity increased from 24 h onwards relative to mock controls. An immunoblot analysis with AtPng1p polyclonal antibody detected a 72-kDa protein whose amount increased at 72 h in TMV-inoculated leaves and in the lesion-enriched areas. A biotin-labeled cadaverine incorporation assay showed that TGase activity occurred in S1 (containing soluble proteins), S2 (proteins released by both cell walls and membranes) and S3 (membrane intrinsic proteins) fractions. In S3 fraction, where changes were the most relevant, TGase activity was enhanced in both mock-inoculated and TMV-inoculated samples, but the stimulation persisted only in the latter case. These data are discussed in the light of a possible role of TGase activity and glutamyl PAs in the defense against a viral plant pathogen.

The negative influence of N-mediated TMV resistance on yield in tobacco: linkage drag versus pleiotropy

Resistance to tobacco mosaic virus (TMV) is controlled by the single dominant gene N in Nicotiana glutinosa L. This gene has been transferred to cultivated tobacco (N. tabacum L.) by interspecific hybridization and backcrossing, but has historically been associated with reduced yields and/or quality in flue-cured tobacco breeding materials. Past researchers have suggested the role of pleiotropy and/or linkage drag effects in this unfavorable relationship. Introduction of the cloned N gene into a TMV-susceptible tobacco genotype (cultivar 'K326') via plant transformation permitted investigation of the relative importance of these possibilities. On average, yield and cash return ($ ha(-1)) of 14 transgenic NN lines of K326 were significantly higher relative to an isoline of K326 carrying N introduced via interspecific hybridization and backcrossing. The negative effects of tissue culture-induced genetic variation confounded comparisons with the TMV-susceptible cultivar, K326, however. Backcrossing the original transgenic lines to non-tissue cultured K326 removed many of these unfavorable effects, and significantly improved their performance for yield and cash return. Comparisons of the 14 corresponding transgenic NN backcross-derived lines with K326 indicated that linkage drag is the main factor contributing to reduced yields in TMV-resistant flue-cured tobacco germplasm. On average, these transgenic lines outyielded the conventionally-developed TMV-resistant K326 isoline by 427 kg ha(-1) (P < 0.05) and generated $1,365 ha(-1) more (P < 0.05). Although transgenic tobacco cultivars are currently not commercially acceptable, breeding strategies designed to reduce the amount of N. glutinosa chromatin linked to N may increase the likelihood of developing high-yielding TMV-resistant flue-cured tobacco cultivars.

A new catalytic activity from tobacco converting 2‐coumaric acid to salicylic aldehyde

Salicylic acid (SA) mediates plant response to pathogen invasion, resulting in hypersensitive response and in the formation of systemic acquired resistance. It is well known that Nicotiana tabacum and other plants respond to Tobacco Mosaic Virus (TMV) infection by increasing the content of SA but the details of SA biosynthesis are still not fully understood. Generally, SA may originate directly from isochorismate (Arabidopsis thaliana), or its C6–C1 skeleton could be synthesized via the phenylpropanoid pathway by β‐oxidation of trans‐cinnamic acid (N. tabacum), 2‐coumaric acid (OCA) (Gaulteria procumbens, Lycopersicum esculentum) or by retro‐aldol reaction of trans‐cinnamoyl‐CoA (Hypericum androsaemum). We report here a novel putative enzyme activity from tobacco, salicylic aldehyde synthase (SAS), catalysing non‐oxidative formation of salicylic aldehyde (SALD) directly from OCA. This chain‐shortening activity is similar to that of 4‐hydroxybenzaldehyde synthase from Vanilla planifolia, Lithospermum erythrorhizon, Daucus carota, Solanum tuberosum and Polyporus hispidus but the enzyme differs in the kinetics of the reaction, substrate specificity and requirements for reducing cofactors. SAS activity is constitutively expressed in healthy tobacco leaves and doubles as a result of infection with TMV. Moreover, the product of SAS activity—SALD, applied exogenously on tobacco leaves, stimulates peroxidase activity and enhances resistance to consecutive infection with TMV. These observations could suggest a contribution of SAS and SALD to the response of tobacco to TMV infection.

Extremely low frequency weak magnetic fields enhance resistance of NN tobacco plants to tobacco mosaic virus and elicit stress‐related biochemical activities

Increasing evidence has accumulated concerning the biological effects of extremely low frequency magnetic fields (ELF-MFs) in different plant models. In the present study, effects of ELF-MFs in tobacco plants reacting to tobacco mosaic virus (TMV) with a hypersensitive response (HR) were evaluated. Plants were exposed for 8 or 24 h (either before or after TMV inoculation) to a static MF, at either -17 or 13 microT, combined with a 10 Hz sinusoidal MF with different intensities (25.6 or 28.9 microT). The working variables were the area and number of hypersensitive lesions in leaves. Following ELF-MFs exposure, an increased resistance was detected, particularly after an 8-h treatment, as shown by the decrease in lesion area and number. Moreover, two enzyme activities involved in resistance mechanisms were analyzed: ornithine decarboxylase (ODC) and phenylalanine ammonia-lyase (PAL). Uninoculated leaves previously exposed to ELF-MFs in general showed a significant increase relative to controls in ODC and PAL activities, in particular for 13 microT static MF plus 28.9 microT, 10 Hz sinusoidal MF (24 h) treatment. In conclusion, ELF-MFs seem to influence the HR of tobacco to TMV, as shown by the increased resistance and changes in ODC and PAL activities, indicating the reliability of the present plant model in the study of bioelectromagnetic interactions.

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.

TRANSGENIC PLANTS EXPRESSING A SINGLE-CHAIN FV ANTIBODY TO TOMATO SPOTTED WILT VIRUS (TSWV) ARE RESISTANT TO TSWV SYSTEMIC INFECTION

Hybridoma cell lines secreting monoclonal antibodies to TSWV were characterized, and one cell line was selected for cloning of the antibody genes. Universal degenerate oligonucleotides were designed to prime PCR amplification of the variable regions of the heavy and light chains of a monoclonal antibody. The PCR products were ligated to produce one single-chain variable fragment (scFv) antibody gene construct that was used to transform Nicotiana tabacum 'Turk'. PCR analyses showed 48% of the 120 regenerated R0 plants contained TSWV scFv genes. The integration and transcription of the scFv gene in the transgenic tobacco plants were confirmed by Southern and northern blot hybridizations, respectively. The expression of TSWV scFv genes in transformed plants was examined by ELISA. TSWV scFv antibodies accumulated to only low levels in transformed plants. Antigen-binding analysis showed that plant-produced TSWV scFv antibodies had the same antigen-binding specificity as the murine TSWV monoclonal antibody. Two TSWV scFv-transformed plants were resistant to TSWV systemic infection as determined by the absence of systemic infection symptoms following TSWV inoculation. The level of virus resistance was correlated with the level of the transgene RNA transcript and scFv antibody production. This resistance was inherited into the R1 generation, and was specific to a lettuce isolate of TSWV (TSWV-L). No resistance was found to Tobacco mosaic virus (TMV) or Cucumber mosaic virus (CMV).

Isolation of a novel Ser/Thr protein kinase gene from oligochitosan-induced tobacco and its role in resistance against tobacco mosaic virus

Oligochitosan induces defense responses to pathogenic microbes in a wide variety of plants by acting as an elicitor. In the present study, mRNA differential display was used to investigate oligochitosan-induced transcriptional activation of defense-related genes. Accordingly, a novel Ser/Thr protein kinase gene was isolated and designated as oligochitosan-induced protein kinase ( oipk). Molecular cloning showed that oipk contains six introns interrupted by seven exons. The open reading frame (ORF) of the gene is 1848 bp, which encodes a putative protein of 615 amino acids with the predicted molecular mass of 70.96 kDa and a p I of 6.32. A plant oipk antisense expression vector was constructed and transformed into tobacco by Agrobacterium tumefaciens. Decreased phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity and decreased resistance to tobacco mosaic virus (TMV) were observed in transgenic tobacco. RT-PCR analysis revealed that oipk was expressed at high levels after oligochitosan induction in wild-type tobacco, but not in transgenic tobacco. These results indicated that oipk is involved in the signal pathway of oligochitosan-induced resistance in tobacco.

Chemical Conjugate TMV−Peptide Bivalent Fusion Vaccines Improve Cellular Immunity and Tumor Protection

Chemical conjugation of CTL peptides to tobacco mosaic virus (TMV) has shown promise as a molecular adjuvant scaffold for augmentation of cellular immune responses to peptide vaccines. This study demonstrates the ease of generating complex multipeptide vaccine formulations using chemical conjugation to TMV for improved vaccine efficacy. We have tested a model foreign antigen target-the chicken ovalbumin-derived CTL peptide (Ova peptide), as well as mouse melanoma-associated CTL epitopes p15e and tyrosinase-related protein 2 (Trp2) peptides that are self-antigen targets. Ova peptide fusions to TMV, as bivalent formulations with peptides encoding additional T-help or cellular uptake via the integrin-receptor binding RGD peptide, showed improved vaccine potency evidenced by significantly enhanced numbers of antigen-reactive T cells measured by in vitro IFNgamma cellular analysis. We measured the biologically relevant outcome of vaccination in protection of mice from EG.7-Ova tumor challenge, which was achieved with only two doses of vaccine ( approximately 600 ng peptide) given without adjuvant. The p15e peptide alone or Trp2 peptide alone, or as a bivalent formulation with T-help or RGD uptake epitopes, was unable to stimulate effective tumor protection. However, a vaccine with both CTL peptides fused together onto TMV generated significantly improved survival. Interestingly, different bivalent vaccine formulations were required to improve vaccine efficacy for Ova or melanoma tumor model systems.

Introgression of a Tombusvirus Resistance Locus fromNicotiana edwardsoniivar. Columbia toN. clevelandii

ABSTRACT A new variety of Nicotiana, N. edwardsonii var. Columbia, was evaluated for its capacity to serve as a new source for virus resistance genes. Columbia was developed from a hybridization between N. glutinosa and N. clevelandii, the same parents used for the formation of the original N. edwardsonii. However, in contrast to the original N. edwardsonii, crosses between Columbia and either of its parents are fertile. Thus, the inheritance of virus resistance genes present in N. glutinosa could be characterized by using Columbia as a bridge plant in crosses with the susceptible parent, N. clevelandii. To determine how virus resistance genes would segregate in interspecific crosses between Columbia and N. clevelandii, we followed the fate of the N gene, a single dominant gene that specifies resistance to Tobacco mosaic virus (TMV). Our genetic evidence indicated that the entire chromosome containing the N gene was introgressed into N. clevelandii to create an addition line, designated N. clevelandii line 19. Although line 19 was homozygous for resistance to TMV, it remained susceptible to Tomato bushy stunt virus (TBSV) and Cauliflower mosaic virus (CaMV) strain W260, indicating that resistance to these viruses must reside on other N. glutinosa chromosomes. We also developed a second addition line, N. clevelandii line 36, which was homozygous for resistance to TBSV. Line 36 was susceptible to TMV and CaMV strain W260, but was resistant to other tombusviruses, including Cucumber necrosis virus, Cymbidium ringspot virus, Lettuce necrotic stunt virus, and Carnation Italian ringspot virus.

Elicitor-Mediated Oligomerization of the Tobacco N Disease Resistance Protein

Plant nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins are similar to the nucleotide binding oligomerization domain (NOD) protein family in their domain structure. It has been suggested that most NOD proteins rely on ligand-mediated oligomerization for function, and we have tested this possibility with the N protein of tobacco (Nicotiana tabacum). The N gene for resistance to Tobacco mosaic virus (TMV) is a member of the Toll-interleukin receptor (TIR)-NBS-LRR class of plant disease resistance (R) genes that recognizes the helicase domain from the TMV replicase. Using transient expression followed by immunoprecipitation, we show that the N protein oligomerizes in the presence of the elicitor. The oligomerization was not affected by silencing Nicotiana benthamiana ENHANCED DISEASE SUSCEPTIBILITY1 and N REQUIREMENT GENE1 cofactors of N-mediated resistance, but it was abolished by a mutation in the P-loop motif. However, loss-of-function mutations in the RNBS-A motif and in the TIR domain retain the ability to oligomerize. From these results, we conclude that oligomerization is an early event in the N-mediated resistance to TMV.

Salicylate Activity. 3. Structure Relationship to Systemic Acquired Resistance

Salicylic acid (2-hydroxybenzoic acid; SA) is a primary signal inducing plant defenses against pathogens. This plant disease resistance, known as systemic acquired resistance (SAR), is an attractive target for the development of new plant protection agents. SAR induction is a multistep process that includes accumulation of pathogenesis-related (PR) proteins. The structure-activity profile of salicylates and related compounds has been evaluated using an inducible PR protein (PR-1a) and plant resistance to tobacco mosaic virus (TMV) as markers. Among the 47 selected monosubstituted and multiple-substituted salicylate derivatives tested, all 8 derivatives that induced more PR-1a protein than SA were fluorinated or chlorinated in the 3- and/or 5-position (3,5-difluorosalicylate > 3-chlorosalicylate > 5-chlorosalicylate > 3,5-dichlorosalicylate > 3-chloro-5-fluorosalicylate > 3-fluorosalicylate > 3-fluoro-5-chlorosalicylate > 3,5-dichloro-6-hydroxysalicylate > SA). In general, substitutions for or on the 2-hydroxyl group or at the 4-position of the ring reduced or eliminated PR-1a protein induction. In contrast, substitutions in positions ortho (3-position) or para (5-position) to the hydroxyl group with electron-withdrawing groups other than chlorine or fluorine decreased induction, and electron-donating groups in these positions also had a deleterious effect on PR-1a induction. PR-1a protein accumulation and reduction in TMV lesion diameter exhibited a log-linear relationship. The seven salicylate derivatives that were the most active TMV resistance inducers were all halogenated in the 3- and/or 5-position (3-chlorosalicylate > 3,5-difluorosalicylate > 3,5-dichloro-6-hydroxysalicylate > 3,5,6-trichlorosalicylate > 5-chlorosalicylate > 5-fluorosalicylate > 3,5-dichlorosalicylate > 4-fluorosalicylate > 3-fluorosalicylate > 3-chloro-5-fluorosalicylate > 4-chlorosalicylate > SA). The correlation between PR-1a protein induction and resistance to TMV confirms the value of using PR-1a induction as a screening tool for developing new plant disease control agents.

Resistance to Tobacco mosaic virus and Botrytis cinerea in tobacco transformed with complementary DNA encoding an inhibitor of viral replication‐like protein

AbstractWe have previously cloned a 1016‐bp‐long complementary DNA (cDNA) clone (named NC330) encoding an inhibitor of virus replication (IVR)‐like protein from induced‐resistant leaves of Nicotiana tabacum cv. Samsun NN. In the present work, NC330 cDNA was cloned in an Agrobacterium binary vector and introduced into the genome of N. tabacum cv. Samsun nn that is susceptible to Tobacco mosaic virus (TMV). Eight R0 were highly resistant, with TMV titres less than 2% of the controls. The progeny of these primary transformants resulted in four TMV‐resistant and four susceptible lines. The progeny of the plant with highest resistance for TMV was selfed during four generations. In the R2 generation, out of 112 plants tested, 3 plants showed excellent resistance and 5 a lesser degree of resistance. NC330 transcripts were detected in these R2 plants as well as in their R1 and R0 parents. Two R2 plants were self‐pollinated to obtain the R3 generation. All the 25 R3 progeny plants of one line contained PCR‐amplifiable NC330, compared with only 6 of the 23 progeny plants from the other line. Following TMV inoculation, virus was undetectable in seven of the eight tested R3 progeny of the first line and five of the six R3 progeny plants of the second line. NC330 transcripts were detected in the TMV‐resistant transgenic plants but also in some susceptible plants. TMV resistance was suppressed when the plants were kept at higher temperatures (32–34°C), whereas returning the plants to lower temperatures (24–26°C) restored resistance. Transgenic plants resistant to TMV were also resistant to B. cinerea following inoculation of seedlings or of whole plants. The presence of the NC330 DNA was correlated with changes in the physiology of the transgenic plant. Seed germination was inhibited at high temperatures and in the dark. Rootlets of germinating seeds of the transgenic plants grew significantly faster than those of control seeds. These observations were correlated with a significantly higher concentration of abscisic acid in the seeds and seedlings of the transgenic plants. In silico analysis suggests that the NC330 protein translated from the NC330 cDNA is a tetratricopeptide repeats protein and that it may be involved in protein–protein interaction as part of the pathogen resistance mechanism.

Rapid determination of methyl salicylate, a plant-signaling compound, in tomato leaves by direct sample introduction and thermal desorption followed by GC-MS

Many plants infested by herbivores or viruses can rapidly produce and accumulate a plant-signaling compound, methyl salicylate (MeSA), in their leaves to activate disease resistance. In the present work, a simple, rapid, and sensitive method was developed for the determination of MeSA in tomato leaves by direct sample introduction and thermal desorption followed by GC-MS. Results show that the proposed method has a low detection limit (0.08 ng mg(-1)) and good precision (RSD = 8.9%). The present method was applied to the investigation of tomato plant defense response to tobacco mosaic virus (TMV) by rapid analysis of volatile compounds in plant leaves. It was found that tomato plants can produce large amounts of MeSA as a defense response to TMV. This indicates that MeSA may be a plant-signaling compound in tomato plant defense response to TMV.

Partial Resistance of Pepper to Bacterial Wilt Is Oligogenic and Stable Under Tropical Conditions.

Genetic analysis of resistance of pepper to bacterial wilt was performed in the doubled haploid progeny from a cross between a resistant parental line PM 687 and a susceptible cultivar Yolo Wonder. After artificial inoculation with a local isolate of Ralstonia solanacearum, the progeny consisting of 90 lines was transplanted into a naturally infested field in Guadeloupe, Lesser Antilles. The 2 years of experimentation resulted in repeatable results, with a high heritability of the resistance, attesting the reliability of the evaluation procedure and the stability of the resistance over years. Two to five genes with additive effects were estimated to control the resistance, indicating an oligogenic control as observed in tomato sources of resistance. Relationships with resistance to other soilborne or tropical diseases were examined. Susceptibility to Tobacco mosaic virus (TMV) and to nematodes (Meloidogyne spp.) were significantly linked with resistance to bacterial wilt, whereas neither resistance to Phytophthora capsici nor to Leveillula taurica were linked. The similarity of the genetics of resistance to bacterial wilt in pepper and tomato and linkage with TMV resistance locus warrant the comparative mapping of the resistance quantitative trait loci in the genomes of the two species.

EDS1 in tomato is required for resistance mediated by TIR‐class R genes and the receptor‐like R gene Ve

In tobacco and other Solanaceae species, the tobacco N gene confers resistance to tobacco mosaic virus (TMV), and leads to induction of standard defense and resistance responses. Here, we report the use of N-transgenic tomato to identify a fast-neutron mutant, sun1-1 (suppressor of N), that is defective in N-mediated resistance. Induction of salicylic acid (SA) and expression of pathogenesis-related (PR) genes, each signatures of systemic acquired resistance, are both dramatically suppressed in sun1-1 plants after TMV treatment compared to wild-type plants. Application of exogenous SA restores PR gene expression, indicating that SUN1 acts upstream of SA. Upon challenge with additional pathogens, we found that the sun1-1 mutation impairs resistance mediated by certain resistance (R) genes, (Bs4, I, and Ve), but not others (Mi-1). In addition, sun1-1 plants exhibit enhanced susceptibility to TMV, as well as to virulent pathogens. sun1-1 has been identified as an EDS1 homolog present on chromosome 6 of tomato. The discovery of enhanced susceptibility in the sun1-1 (Le_eds1-1) mutant plant, which contrasts to reports in Nicotiana benthamiana using virus-induced gene silencing, provides evidence that the intersection of R gene-mediated pathways with general resistance pathways is conserved in a Solanaceous species. In tomato, EDS1 is important for mediating resistance to a broad range of pathogens (viral, bacterial, and fungal pathogens), yet shows specificity in the class of R genes that it affects (TIR-NBS-LRR as opposed to CC-NBS-LRR). In addition, a requirement for EDS1 for Ve-mediated resistance in tomato exposes that the receptor-like R gene class may also require EDS1.

Members of 14-3-3 protein isoforms interacting with the resistance gene product N and the elicitor of Tobacco mosaic virus

A functional cDNA for the resistance gene N from Nicotiana tabacum cv. Samsun NN to Tobacco mosaic virus (TMV) was obtained by reverse transcription polymerase chain reaction. Recombinant proteins containing various domains of the N protein were used in far-Western screening for the N-interacting protein from tobacco cDNA expression libraries. Nucleotide sequence analysis revealed that 199 cDNAs of 217 positive clones screened from 1.6 × 106 phage plaques were related to the 14-3-3 gene family. Phylogenetic analysis indicated that the 14-3-3 proteins were divided into 17 different 14-3-3 isoforms, including hitherto unidentified novel isoforms i-1 and i-2. In vitro binding assays showed that the probes TIR and LRR domains of N protein and helicase (HEL) domain of TMV replicase bound significantly to 14-3-3 isoforms of groups ω and ψ. These domains barely bound to those of group κ and did not bind to that of group e. The same probes did not bind to any domains of N protein or the viral HEL domain. Northern blotting showed an increase in 14-3-3 isoform h transcripts coinciding with PR-1a induction after TMV inoculation. The 14-3-3 isoform h as a probe had significant binding to the LRR domain of N protein and the viral HEL domain, but there was only minor binding to the TIR domain. These results raise the possibility that 14-3-3 acts as a scaffolding or adaptor between the N protein and the helicase domain to support the interaction between the R gene product and the viral elicitor.

Involvement of MEK1 MAPKK, NTF6 MAPK, WRKY/MYB transcription factors, COI1 and CTR1 in N-mediated resistance to tobacco mosaic virus.

The tobacco N gene, a member of the Toll-interleukin 1 homology region/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class of resistance (R) genes, confers resistance to tobacco mosaic virus (TMV). We used a candidate gene approach to identify known defense genes that were also involved in N signaling. The requirement for these genes was determined by downregulating their expression using the well-established tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS). Silencing of genes encoding a mitogen-activated protein kinase (MAPK) NTF6/NRK1, or an MAPK kinase (MAPKK) MEK1/NQK1, attenuated N-mediated resistance to TMV. We also found that N resistance is compromised in plants in which expression of WRKY1-WRKY3 and MYB1 transcription factors were downregulated. In addition, suppression of jasmonic acid (JA) signaling component COI1 ortholog affected N function. However, downregulation of expression of CTR1 ortholog leads to more rapid hypersensitive response (HR). The involvement of these genes in N- and other R-gene-mediated defense provides further evidence for the convergence of downstream signaling pathways of different R genes.

Investigation of Tomato Plant Defence Response to Tobacco Mosaic Virus by Determination of Methyl Salicylate with SPME-Capillary GC-MS

Headspace SPME was applied to investigation of tomato plant defence response to tobacco mosaic virus (TMV) by determination of compounds emitted from tomato plants at the extraction conditions of 25 °C and 15 min. It was found that TMV-inoculated tomato plant released large amount of methyl salicylate (MeSA) as response to TMV and MeSA concentration changed dramatically with time after inoculation. Gaseous MeSA as a signaling compound could induce in surrounding tomato plants to produce salicylic acid (SA) and synthesize and release MeSA. These results show that MeSA might be an airborne plant-signalling molecule in tomato plant response to TMV. The present method provided low detection limit of 2.0 ng L−1 and needed little sample preparation time (15 min), so the method makes it easy to find the critical times of tomato plant response to TMV by fast determination of MeSA released from tomato plant.

Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance.

Nicotiana tabacum Togt encodes a scopoletin glucosyltransferase (UDPglucose:scopoletin O -beta-D-glucosyltrans- ferase, EC 2.4.1.128) known to act in vitro on many different substrates including the 6-methoxy-7-hydroxy- coumarin scopoletin. This phenolic compound accumulates in vast amounts, essentially in its glucosylated form scopolin, in tobacco during the hypersensitive response (HR) to tobacco mosaic virus (TMV). To identify the physiological role of this pathogen-inducible UDP-Glc glucosyltransferase (UGT), we generated TOGT over-expressing transgenic plants. Although no endogenous scopoletin or scopolin could be detected before infection, the accumulation of both the aglycone and the glucoside was found to be 2-fold higher in transgenic plants after inoculation with TMV than in wild-type plants. Scopoletin UGT activity in plants over-expressing Togt was significantly higher during the HR than in control plants. This up-regulated activity was associated with a strong increase of the bright blue fluorescence surrounding the HR-necrotic lesions under UV light, which is known to correlate with scopoletin and scopolin abundance. Necrosis appeared sooner in transgenic plants and lesions developed faster, suggesting an accelerated HR. Unexpectedly, the viral content in each lesion was not significantly different in transgenic and in wild-type plants. These results are discussed in relation to the role of TOGT as the major UDP-Glc: scopoletin glucosyltransferase and to the importance of scopoletin accumulation during the HR.

Identification of Tomato mosaic virus Infection in Lisianthus in Taiwan

In recent years, Lisianthus (Eustoma russellianum (Don.) Griseb) has become popular as potted plants and cut flowers in Taiwan. They are grown in the central and southern regions of the island. Since 1998, diseased plants with mosaic symptoms, followed by necrosis of leaf tissues, were observed in commercial greenhouses and field-grown lisianthus. Newly emergent leaves were curled and smaller compared with those on healthy plants. These symptoms greatly decreased the commercial value of the crop. Rigid rods similar to tobamoviruses that measured 300 × 18 nm were found consistently associated with symptomatic plants. In July 2002, a virus culture was isolated from diseased lisianthus from Chiayi County, Taiwan and established and maintained in systemic hosts Nicotiana tabacum L. and N. benthamiana Domin. Chlorotic and necrotic spots developed on lisianthus leaves 1 to 2 weeks after inoculation with the virus; symptoms eventually became systemic. Virions were purified from inoculated N. tabacum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the virus contained one 18-kDa (Mr) polypeptide. The virus reacted positively in agar gel double diffusion tests and enzyme-linked immunosorbent assays with antisera prepared to Tobacco mosaic virus (TMV) or Tomato mosaic virus (ToMV) (gifts of S. D. Yeh, National Chung Hsing University, Taichung, Taiwan). A viral coat protein (CP) gene approximately 0.5 kb was amplified by reverse transcription-polymerase chain reaction from total RNA prepared from infected N. benthamiana using 5'-GCGAGCCATGGATTCTTACTCAATTACT as a forward primer and 5'-ACTCTCGGATCCTTAAGATGCAGGTGCAGA as a reverse primer. Comparison of the 480-nt CP gene region with that of ToMV-OM (GenBank Accession No. X02144) (3) revealed 99.2% nucleotide identity and 99.4% amino acid identity. It shares, however, 74.4% nucleotide identity and 83.9% amino acid identity with CP genes of TMV-U1 (GenBank Accession No. AX040174) and TMV-vulgare (GenBank Accession No. J02415) (1). The virus induced local lesion responses similar to ToMV on inoculated N. tabacum cv. White Burley, N. sylvestris Speg. & Comes, and Datura stramonium L. Inoculation of TMV, however, resulted in a systemic infection in these plants. Results from sequence analysis and diagnosis based on host reaction to the virus inoculation indicated that the tobamovirus infecting lisianthus in Taiwan is an isolate of ToMV. The virus is economically important to lisianthus and tomato in Taiwan. To our knowledge, this is the first report of ToMV causing disease on lisianthus in Taiwan. The disease was previously observed on lisianthus in Italy (2).