Tobacco mosaic virus (TMV) and Tobacco mild green mosaic virus (TMGMV) are both known to induce hypersensitive response (HR) local lesions in Nicotiana megalosiphon, a hybrid plant from a cross between N. suaveolens and N. fragrans, but only TMV is capable of systemic movement. Therefore, the determinants of induction of hypersensitive response and systemic movement of TMV and TMGMV in N. megalosiphon were further analyzed. HR was shown to be independent of the temperature indicating that the resistance responses are different from N gene responses induced in tobacco (N. tabacum cv. Xanthi-nc.) to TMV. Comparison of lesion growth between wild-type and transgenic tobacco plants encoding salicylate hydroxylase (nahG) indicated that TMV spread similarly in N. megalosiphon and Xanthi-nc./nahG plants. In addition, exogenous application of SA did not prevent systemic movement of TMV. Coat protein-deficient TMV failed to induce HR and move systemically in N. megalosiphon indicating that CP is the inducer of HR and determinant for systemic movement. However, single epidermal cells expressing TMV-CP did not undergo cell death suggesting that formation of HR cell death requires viral movement out of the initially infected epidermal cells or the presence of intact virus particles. Furthermore, comparison of different TMV strains, including virus vector carrying the CP of TMGMV-U5 in place of TMV-CP, showed no differences in the induction of HR and timing of systemic virus movement suggesting that CP is not the determinant for differential invasion of TMV and TMGMV.
Keywords: Tobacco mosaic virus; Tobacco mild green mosaic virus; Hypersensitive response; Systemic virus movement; Coat protein
Tobacco Mosaic Virus (TMV) is the type species of the Tobamovirus genus within Alphavirus-like superfamily of viruses. There are several other highly homologous tobamoviruses that are either species in Tobamovirus genus or strains of TMV. Positive-sense, single-stranded RNA genome (6400 bp) of TMV encodes four proteins; two for replication with read-through stop codon, one for movement (MP) and one structural or Coat Protein (CP). All viral proteins possess several functions in virus life cycle. Replicase proteins function in virus replication while MP was the first viral protein shown to mediate cell-cell transport [1,2]. TMV-MP has also been shown to enhance RNA silencing . CP forms the virus particle and mediates systemic movement of the virus, but is also know to induce hypersensitive cell death response (HR) in some host plants similarly to replicase and MP proteins. For example, HR is elicited by the TMV-CP in Nicotiana sylvestris plants carrying the N´ gene [4-6] and in pepper carrying the L2 or L3 genes [7,8]. The replicase protein of TMV induces HR in tobacco (N. tabacum cv. Xanthi-nc.) plants containing the N gene , and the movement protein (MP) of Tomato mosaic virus induces HR in tomato plants harbouring the Tm-2 or Tm-22 gene . The exact mechanisms how viral proteins and host resistance gene products interact to cause disease symptoms are mainly unknown. In addition, there are many other plant viral proteins (from other virus groups) that are known to elicit the HR in various plant species but in most cases, the plant resistance gene(s) involved in the necrotic response or the mechanisms and mediators of the resistance response have not been determined [11,12].
The cascade of events leading to defense response involves various aspects that are dependent on the plant´s ability to respond to the structure and the amount of the inducer produced by the virus and the viruses´ ability to escape these defense responses by moving ahead of or by suppressing them. Oxidative burst has been shown to play a role in the early phase of TMV infection, and it has been suggested that specific epitopes within TMV-CP define the specificity of that response . Another important mediator of defense responses is Salicylic Acid (SA), which has been shown to increase around the TMV-induced HR lesions in resistant tobacco plants . TMV-induced lesions have been shown to be larger in the transgenic tobacco plants that express the bacterial salicylate hydroxylase (nahG), which converts free SA to catechol [15,16] suggesting that SA acts directly against the lesion growth. Induction of salicylic acid during incompatible plant-pathogen interaction is apparently a universal phenomenon, and there are only few reports demonstrating that such responses are SA-independent . SA is also known to interfere with the replication of TMV . The expression levels of the TMV-MP have been suggested to correlate with the size of the virus-induced lesions [18,19]. Yet, data has also been shown that the size of lesions induced by TMV is independent of the amount of MP .
¨Previously, it has been shown that TMV-U1 and Tobaco mild green mosaic virus (TMGMV-U5 have differential invasion of N. megalosiphon, a hybrid plant from a cross between N. suaveolens and N. fragrans . Both TMV and TMGMV induced necrotic local lesions (HR) in inoculated leaves of N. suaveolens but only TMV was capable of systemic movement and induction of systemic HR (SHR) . In the present study, further analyses of defense responses induced in N. suaveolens by TMV were performed. It will be demonstrated that HR and SHR are independent of temperature and the amount of the virus. Salicylic acid-induced responses were analyzed by using nahGplants and by application of exogenous SA, and shown not to interfere with systemic movement of TMV. Coat protein-deficient TMV did not induce lesions indicating that CP is the inducer of HR. In addition, TMV bearing the coat protein of TMGMV-U5 moved systemically in N. suaveolens indicating that although CP is essential for systemic movement of TMV within N. megalosiphon, it is not the determinant for differential invasion of TMV and TMGMV in N. megalosiphon.
Viruses and plants
Tobacco mosaic virus (genus Tobamovirus) strain U1 (TMV-U1; type strain), TMV strain U2 (currently known as Tobacco mild green mosaic virus, TMGMV-U2; , Flavum [24,25], TMV-CP(-) (coat protein-deficient TMV;  and TMV-30B (TMV-U1-derived viral gene expression vector with coat protein from TMGMV-U5;) are laboratory strains that have previously been described; TMGMV is a designation of a group of tobamovirus isolates that are serologically similar. Strain U2 was originally found as a component of U1 population  whereas U5 designated a virus isolated from N. glauca . Nicotiana tabacum cv. Xanthi-nc. (necrotic host for tobamoviruses except the Obuda strain) and Nicotiana megalosiphon (obtained from prof. V.A. Sisson; North Carolina State University, USA) were used as host plants. N. suaveolens is a species native to the Australian continent, and a hybrid form of a cross between N. suaveolens (n = 16) and N. fragrans (n = 24) . It belongs to a subgenus Nicotiana, and has no known species with known resistance gene(s). Transgenic nahG plants (created in the background of N. tabacum cv. Xanthi-nc.;  encode salicylate hydroxylate that hydrolyzes salicylic acid.
Plant growth and virus inoculations
Inoculated plants were kept in a greenhouse at 22-25oC temperature with a 16-h photoperiod or as indicated. Primary leaves of 4 to 7 weeks old Nicotiana megalosiphon were mechanically inoculated with Carborundum as abrasive. Temperature-dependence of HR in N. suaveolens was tested by shifting TMV-inoculated plants to +32oC. Lesion size was measured by ocular micrometer and in average, 30 lesions in four individual leaves were counted in 3-4 independent sets of experiments. Virus titer was adjusted appropriately by serial dilution when counting individual lesions. Normally, dilution resulting in 10 lesions per leaf was used when measuring the size of lesions (to avoid overlapping regions). Lesion growth was followed for up to 7 days. The movement of the virus to the upper, un-inoculated leaves was recorded at days post-inoculation (d p.i).
Treatments with salicylic acid
Typical SAR assays by performing primary and secondary inoculations at 7-day intervals could not be performed because all tobamovirus species or strains used in the work, spread systemically and induced systemic HR in upper leaves within 6-12 days. Therefore, plants were hydroponically fed by placing entire plants into the soil watered by a solution containing 2 mM of salicylic acid (SA) three days prior to inoculation with TMV . Watering with SA solution was continued throughout the experiment. The local and systemic spread of viruses was monitored.
In vitro transcription of coat protein-deficient TMV
The coat protein-deficient derivative of TMV-U1, TMV-CP(-) , which was subcloned under T7 promoter, was linearized with KpnI and subjected to in vitro transcription using T7 RNA polymerase with CAP analog. In vitro transcripts were inoculated onto N. tabacum cv. Xanthi-nc. and N. suaveolens .
Cloning of coat protein of TMV-U1 in fusion to GFP into pRT101 vector
Plasmid pTMV304 (cDNA copy of TMV-U1) and was used as a template in a PCR reaction for cloning of TMV-U1 coat protein. The PCR product of about 480 bp was cloned in fusion to GFP into pRT101-vector digested with EcoRI and XbaI under the 35S promoter. The resultant clone, pRT101-CP-GFP was used in transient biolistic assay.
Particle bombardment and microscopy
Mature 4-6-week-old leaves were subjected to particle bombardment using a PDS-1000/He Biolistic Particle delivery System (Bio-Rad, Hecules, CA). Plasmids destined for particle bombardment were purified with the Bio-Rad Midi-kit. Coating of tungsten micro carrier particels (1.3 μm; Bio-Rad) with plasmid DNA was according to the manufacturer´s instructions. Bombarded leaves were incubated at 20oC in the dark on prewetted filter paper in parafilm-sealed Petri dishes prior to microscopic analysis of GFP expression.
The confocal laser scanning microscope was performed using Zeiss Axiovert 10 inverted microscope as previously described . In short, the 488 nm argon-ion laser was used for excitation of GFP fluorescence. The emitted fluorescence was filtered with 500-530 nm bandpass filter. Optical sections were acquired with a Zeiss, Apochromat 40 x 1.2 NA water immersion objective. Image processing was performed using Zeiss LSM510 Meta program and Adobe Photoshop CS3 package.
Detection of TMV coat protein by western blot
Leaf material was disrupted in liquid nitrogen, suspended in SDSsolubilization buffer, and separated on SDS-PAGE gels . Proteins were transferred to Polyvinylidene fluoride membranes (Millipore, Bedford, USA). CP was detected using an anti-TMV antibody as primary antibody and horseradish peroxidase conjugate as secondary antibody (Enhanced chemiluminescence Western blot detection system, Amersham, Buckinghamshire, U.K.).
Number of infection sites and temperature do not affect systemic movement and induction of Systemic Hypersensitive Response (SHR) by Tobacco mosaic virus in N. megalosiphon. The number of infection sites defines the number of cells that respond to the virus, and mediate the systemic resistance responses. Previously, it has been shown that TMV-U1 but not TMGMV-U5 moves systemically and induces Systemic Necrosis/Hypersensitive Response (SHR) in N. megalosiphon  but it was not shown whether the number of infection sites affected systemic movement of TMV-U1. It was, therefore, analysed whether systemic virus spread correlated temporally with the number of lesions in primary leaves. Virus preparation was serially diluted and inoculated onto primary leaves of 4-week old plants, and virus spread was followed on daily basis (Table 1). Systemic movement of TMV occurred at the same time in most plants, except at virus dilution where only 1 lesion formed in primary leaf. In such cases, however, the systemic movement and formation of SHR were delayed only by a few days. Tobacco plants (N. tabacum cv. Xanthi nc.) were used as control, and no SHR was detected in tobacco as expected (Figure 1a). Systemic symptoms induced by TMV in N. suaveolens were first observed at 4-7 d p.i. Their formation was dependent on the size of the plant and preceded the systemic necrosis, which appeared within 6-11 d p.i. (Table 1). That is N. suaveolens plants were systemically infected by TMV before the onset of HR in those cells/tissues invaded by TMV. This correlated with the number of initial infection sites.
|Dilution factor||Lesions number in primary leaf||Systemic necrosis (d p.i.)‡|
*Plants with a single lesion were used in the analyses.
‡Data represents average of three consequtive experiments with 10 plants in each experiment.
Table 1: Effect of infection sites (lesion number) on systemic movement of Tobacco mosaic virus in Nicotiana megalosiphon.
Figure 1: Local and systemic movement and induction of hypersensitive responses of Tobacco mosaic virus (TMV) in tobacco plants (N. tabacum cv. Xanthi nc.) (A) and N. megalosiphon (B-D). Block arrows indicate the size of a single, growing lesion towards the main vein at 5 d p.i. (C) and 12 d p.i. (D).
The spreading of local lesions initiated from a single lesion was also followed on daily basis. TMV moved in continuous cell-cell mode reaching and necrotizing main veins (Figure 1c and 1d). Interestingly, similar continuous cell-cell movement has previously been shown in tobacco plants compromised with salicylic acid production (in plants expressing the product of nahG gene; salicylate hydroxylase) . In contrast, in wild-type tobacco (N. tabacum Xanthi-nc.) plants, lesion growth is ceased by day 7 p.i. before reaching veins . This indicated that the possible induction of the resistance responses by high titer virus or lack of infection sites in inoculated leaves did not significantly limit the systemic spread of TMV to upper, non-inoculated leaves of N. suaveolens . The data also suggest that the defense responses initiated in N. suaveolens against TMV are compromised.
Effect of salicylic acid on lesion growth and systemic movement of TMV in N. suaveolens
Previously, it was shown that the timing and kinetics of local lesion growth of TMV and TMGMV in N. suaveolens was similar . However, as indicated above the TMV-induced lesion grew in continuous cell-cell manner indicating that cell-cell movement of TMV was not efficiently restricted. Therefore, cell-cell movement of TMV in N. suaveolens was compared to the movement in wild-type and transgenic nahG tobacco plants. The results indicate that TMV moves at similar rate in N. suaveolens as in the nahG plants in the early phase of infection (3 d p.i.) (Table 2). However, in later phases (7 d p.i.) the difference in lesion size between tobacco and N. suaveolens but not between nahG and N. suaveolens plants became statistically significant (P < 0.0005; (Table 2) indicating that TMV was capable of moving further in N. suaveolens than it did in tobacco plants. This suggested that salicylic acid (SA) or SA-mediated resistance responses are not effectively induced by TMV in N. suaveolens and do not prevent systemic spread of the TMV.
|Plant||Lesion size (mm)a|
|3 dpi||7 dpi|
|Xanthi-nc.||2.38 ± 0.37b||4.54 ± 0.61c|
|nahG||2.56 ± 0.44||5.73 ± 0.73d|
|N. megalosiphon||2.63 ± 0.50b||5.38 ± 0.83c,d|
aLesion size is the mean of 30 lesions on four plants (one leaf per plant) ± SD. bdP > 0.05. cP<0.0005. dP>0.05. Experiment was repeated four times with similar results.
Table 2: Size of local lesions after TMV infection in tobacco (N. tabacum cv. Xanthi-nc.), transgenic tobacco expressing salicylate hydroxylase (nahG), and in N. megalosiphon.
As TMV moved fast both locally and systemically leading to full necrosis of the leaf, traditional half-leaf local lesion assay could not be performed to analyze the induction of the systemic acquired resistance (SAR). Instead, to analyze the dependency of resistance response on salicylic acid (SA), the key component in the pathway leading to SA, potted plants were fed with salicylic acid. Plants were watered with 2 mM SA-solution three days prior to inoculation with TMV using virus dilution that gave separate lesions (10 per leaf), and lesion size was measured at 3 and 5 d p.i. (Table 3). Although, the difference between the lesions in water and SA-treated leaves was statistically significant (Table 3, P<0.005), TMV was capable of spreading systemically in N. suaveolens (data not shown) leading to SHR (as in Fig. 1b). This indicated that the resistance mechanism in N. suaveolens either does not respond to SA-treatment similarly to tobacco plants bearing N gene for resistance, or that the SA induction leads only to weak responses, which do not restrict virus spread in N. suaveolens .
|Treatment||Lesion size (mm)a|
|3 dpib||5 dpib|
|water||1.97 ±0.63||3.01 ±0.56|
|salicylic acid||1.43 ±0.55||2.68 ±0.31|
aLesion size is the mean of 30 lesions on four plants (one leaf per plant) ±SD. bP < 0.0005. Experiment was repeated four times with similar results.
Table 3: Effect of exogenous application of salicylic acid on lesion growth in N. megalosiphon. TMV was inoculated at dilution that resulted in 10 lesions per leaf.
Resistance to TMV is independent of temperature
To test the temperature-sensitive nature of the resistance response against TMV in Nicotiana megalosiphon, plants were inoculated with TMV-U1, and kept at 32oC for 2 days. Within that time, local lesions formed in lower, inoculated leaves of N. suaveolens (Figure 2a), but not in Xanthi-nc (Figure 2b), and the virus also moved systemically inducing the SHR (data not shown). The temporal formation of SHR was at least twice as fast as in the case of TMV at the same temperature (data not shown). This implied that the pathway leading to HR in N. suaveolens is temperature-independent unlike the HR induced by TMV in resistant tobacco plants (Figure 3b) , or that resistance function downstream of N gene . Virus induced local lesions also at the lower temperature (17°C), and moved systemically but in slower rate implying that there is linear effect of temperature on systemic movement but not induction on HR or SHR (data not shown).
Induction of hypersensitive response in N. suaveolens by viral coat protein
Since the resistance response was not dependent on temperature, it is unlikely that it is mediated by N gene-like responses. As N gene is inducible by TMV replicase, we analyzed whether viral capsid is the inducer of HR as is the case of N’ gene-mediated responses. TMV mutant, TMV-CP (-), which is deficient of coat protein, was used in in vitro transcription assay, and the transcripts were inoculated onto plant leaves. TMV-CP (-) is known to move cell-cell but not systemically in tobacco plants . As a control, tobacco plants in which viral replicase is the inducer of HR, were inoculated with TMV-CP (-). Local lesions were visible in tobacco but not in N. suaveolens leaves indicating that infection with TMV-CP (-) transcripts was successful and that CP is essential for HR response in N. suaveolens (Figure 3). No virus infectivity was recovered from upper leaves indicating that TMV-CP(-) did not move systemically (data not shown). The data suggest that CP is the determinant for systemic movement also in N. suaveolens similarly to tobacco plants.
As the CP was the apparent inducer of HR in N. suaveolens , its ability to induce HR in single epidermal cells of N. suaveolens was also analyzed. When CP fused to green fluorescent protein was introduced into epidermal cells by biolistic means, CP-GFP was localized to cytosol and the cells were fluorescing after 2 days of bombardment (Figure 4a). To verify expression of CP-GFP, the fusion protein was extracted from bombarded leaves and detected by TMVspecific antiserum (Figure 4b). This indicated that cell death does not occur in individual, epidermal cells of N. suaveolens or that a certain threshold concentration of coat protein or intact virus particles are needed to induce HR.
Figure 4: Particle bombardment of coat protein into epidermal cells. Coat protein was cloned in fusion to green fluorescent protein into pRT101- transient expression plasmid under control of CaMV 35S promoter and introduced into epidermal cells by biolistic means (A). Coat protein was extracted from bombarded leaves and detected using TMV-specific antiserum (B). M. Prestained Protein Marker, Broad Range (New England BioLabs, UK), 1. wild-type tobacco, 2. CP-GFP from bombarded leaves.
Coat protein is not the determinant for differential invasion of Nicotiana megalosiphon by TMV and TMGMV. Although, TMV-CP is the likely inducer of HR in N. suaveolens (Figure 4), TMV-CP (-) could not move systemically in it indicating that CP is also the determinant for systemic movement of TMV in N. suaveolens (data not shown). Previously, it has been demonstrated that both TMV and TMGMV (strain U5) induced local lesions but only TMV was capable of spreading systemically in N. suaveolens . In the current study, TMV-U1, TMGMV-U2, Flavum and TMV-30B (a modified TMV-U1- based virus vector bearing CP from TMGMV-U5;  were tested for the induction of HR local lesions, its ability to move systemically and to induce SHR . All viruses, including TMV-30B, went systemic and induced SHR in N. suaveolens . The timing of systemic spreading did not differ significantly between the viruses (Table 4). Flavum appeared to be faster in systemic movement than the other viruses but the reason for such behaviour is not known. Since TMV-U1 and TMV-30B coding TMGMV-U5-CP moved systemically, the difference in systemic movement of TMV-U1 and TMGMV-U5  in N. suaveolens is not due to the CP.
|TMV strain||Inoculated leaves||Upper leaves|
Experiments were repeated three times with 3 plants in each experiment for each virus
Table 4: Infectivity and symptoms in Nicotiana megalosiphon induced by tobamo virus strains. Timing of the necrotic symptom appearance is shown as days post-inoculation (d p.i.).
The winner in a battle between a plant and an invading viral pathogen is often dependent on how quickly the virus can proliferate, move and cause damage, compared to how fast the plant can respond and prevent the virus from causing damage. The resistance response in virus-plant interaction is normally due to a Resistance (R) gene, and many plant species encode R genes that can recognize specific elicitor or Avirulence (avr) factors. Several natural resistance genes have been characterized  but apparently many more remain unidentified. The induction of the HR is a race-specific event between R gene and Avirulence (Avr) gene products, and has been implicated in restriction the spread of pathogen in infected tissue leading hypersensitive cell death responses (HR); this is often exemplified by formation necrotic local lesions at the site infection . However, HR is not always prerequisite for formation of R-gene-mediated resistance responses [38-40], and, therefore, other factors must contribute to the localization of the virus . In most cases R-avr interaction also leads to formation of systemic acquired resistance (SAR) in upper, non-inoculated leaves. Occasionally, efficient virus spread is accompanied by systemic HR (SHR;  that follows virus movement.
The induction of HR and systemic movement of Tobacco mosaic virus (TMV-U1) and Tomato mild green mosaic virus (strain U5) in N. suaveolens have previously been compared ; TMV moved systemically inducing systemic HR (SHR) while TMGMV was localized to inoculated leaves. In the current study, the defense responses induced in N. suaveolens against TMV were analyzed further. Incubating N. suaveolens plants in different temperatures for 3-5 days always resulted in lesion formation and did not prevent formation of SHR. Thus, temperature had no effect on formation of HR in N. suaveolens . Previously, it has been shown that TMV infection in tobacco plants bearing N gene is temperature-sensitive  indicating that the defense responses to TMV are not similar between resistant tobacco and N. suaveolens .
Lesion growth in TMV and TMGMV-U5 inoculated leaves was followed for 8 days showing only a slight difference between N. suaveolens and N. tabacum cv. Xanthi-nc. . The differential invasion of TMV and TMGMV in N. suaveolens was explained by increased TMV accumulation around lesions and TMV replication in protoplasts compared to TMGMV. However, comparison of replication levels cannot be used in the context of HR since HR does not occur in protoplasts . Therefore, the effect of the amount of virus on virus movement and formation of SHR was analyzed by diluting the virus preparation down to a single lesion; systemic virus spread and SHR occurred even from a single inoculation point within a leaf. Interestingly, TMV infection initiated from a single lesion continued at least for 2 weeks, which is different to TMV in tobacco plants where lesion growth ceases at 7 d p.i. and never reach the main veins . This implied that the formation of resistance responses was compromised. One explanation for systemic movement of TMV inspite of HR could be delayed occurrence of biochemical and physiological events that are associated with HR and Systemic Acquired Resistance (SAR). Such events usually involve induction of Salicylic Acid (SA), PR-proteins, reactive oxygen species and cell wall lignification. In transgenic plants expressing baculovirus p35 protein, which inhibits caspases , HR was delayed and the virus was capable of spreading beyond the initial infection site, and systemically infect the plant. For tobamovirus mutants in plant hosts bearing N´ or Tm-2 resistance genes [10,43], ability to overcome HR and spread systemically may also be related to delayed HR.
As the TMV-induced lesion growth in N. suaveolens was similar to the situation where SA-mediated defense responses were delayed leading to viral spread in a continuous cell-cell manner , it is possible that the resistance pathway induced by TMV does not involve or is independent of salicylic acid. In the current study it was shown that TMV-induced lesions grew unrestricted in inoculated leaves followed by symptoms of systemic HR (SHR) within 3-6 days in N. suaveolens whereas in N. tabacum cv. Xanthi-nc. lesion growth in inoculated leaves was confined to the inoculated leaf. It is of interest that in the early stage (3 d p.i.) of lesion growth TMV spread equally in both tobacco, nahG and N. suaveolens but in the later stage (> 7 d p.i.), the virus movement was statistically slower in tobacco compared to nahG and N. suaveolens plants. This indicates that HR is either delayed and TMV is capable of moving ahead of defense responses, TMV is capable of restricting the formation of resistance responses, or simply that resistance response is not strongly induced in N. suaveolens . Previously, it was demonstrated that interference in N-gene mediated resistance pathway delayed HR formation and lead to SHR but at much slower rate than described here (6 vs. 10 days) . They suggested that SHR can be the cause of two independent actions; the interference in the expression/splicing of resistance gene that may delay the onset of the HR and allow the virus to spread, or the ability of the virus to spread in the tissue beyond the systemic signals that would prevent virus movement. Further analysis of the effects of SA in N. suaveolens were performed by exogenous feeding of SA followed by TMV infection. In contrast to TMV in tobacco plants, SA application had only slight effect on the lesion formation in N. suaveolens . It has been shown previously that SA accumulates around the lesions and either inhibits the replication of the virus at the point of infection or delays virus movement out of inoculated tissue . TMV was shown to replicate efficiently in N. suaveolens compared to TMGMV  suggesting that SA does not act effectively on TMV. Since exogenous application also did not have an effect on systemic movement of TMV, other (viral) factors must contribute to the success of the virus to invade N. suaveolens .
Several plant viral proteins are involved in the induction of cell death via hypersensitive response-like reaction. Viral coat protein has been involved in HR in PeMV-infected pepper carrying the L3 or L2 genes [7,8], in Potato virus X (PVX) -infected potato plants that have the Nx gene , and also in TMV-infected Nicotiana sylvestris plants carrying the N´ gene [4-6]. HR is also induced by the replicase protein of TMV in N. tabacum plants containing the N gene  and by the Movement Protein (MP) of Tomato mosaic virus in tomato plants harbouring the Tm-2 or Tm-22 gene . Using the coat proteindeficient mutant, it was shown here that the CP is also determinant for HR in N. suaveolens and determinant for the systemic movement, since it could not infect plants systemically. In contrast, when plasmids carrying CP gene under the 35S promoter were biolistically introduced into epidermal cells, no cell death was detected based on the fluorescence after 2 days. This implied that HR is not induced in single cells as shown previously also for Cucumber mosaic virus in Chenopodium amaranticolor and TMV in tobacco, respectively [45,46], or that threshold level of CP or intact virus particles are needed for induction of cell death.
CP-mediated resistance response has been shown to depend on the structure of the CP . Thus, the type of defense responses formed in N. suaveolens to TMV is possibly also affected by CP structure. However, several tobamoviruses with different CPs (TMV-U1, TMGMV-U2, Flavum and TMV-30B with U5-CP) were shown to induce HR local lesions in inoculated leaves and SHR in N. suaveolens indicating that they all are efficient inducers of HR. Previously, it was shown that TMGMV-U5 does not spread systemically in N. suaveolens . As the infection with TMV-30B (encoding CP of TMGMV-U5 in the place of its own CP;  led to formation of systemic HR (SHR), the viral determinant for differential invasion of TMV and TMGMV is not likely to be the coat protein, although it is the determinant for systemic movement as shown before for TMV in tobacco . Inoculation of resistant Nicotiana tabacum cv. Xanthi leaves with mutant virus in which the MP of TMGMV-U5 was transferred into the genome of TMV led to smaller lesions than inoculation with TMV . This suggested that the size and growth rate of necrotic lesions on N. tabacum Xanthi-nc. was influenced by the MP. Although, it has been shown that there may be limitations in cell-cell movement in resistant host [19,48], there was no difference in the lesion size between TMV and TMGMV in N. suaveolens  indicating that the rate of cell-cell movement was not affecting the systemic virus movement. TMV replicase also has a role in systemic spread of TMV in tobacco plants , and its possible role in the lack of systemic spread of TMGMV in N. suaveolens should not be excluded particularly since TMGMV replicated less efficiently than TMV. Identification of the protein determinant for systemic movement with subsequent sequence comparisons would allow identification of specific domain(s) required for systemic movement of tobamoviruses in N. suaveolens .
Finally, it is intriguing to speculate of the nature of the resistance in N. suaveolens . Various plant viral genes from different taxonomic groups are known to induce resistance responses against specific R-gene , and all TMV-related proteins induce HR in specific host plants. An N gene-associated, but temperature-independent resistance to TMV that is not mediated by SA has been demonstrated . This resistance was abolished by CMV-1a protein suggesting that it may interact with putative resistance factor. However, since the resistance response was induced by CP, it is unlikely to be linked to responses occurring in N. suaveolens . Instead, these responses are similar to TMV response in N. sylvestris bearing N´ gene; N´ gene-mediated responses are inducible by CP, and all tobamovirus strains except the U1-strain induce HR in it, and many are also capable of induction of SHR . It has also been shown  that CaMV gene VI elicits SHR by the interaction of a single gene in N. clevelandii using interspecific cross between N. clevelandii and N. bigelovii, which were fertile . However, no virus strains with different behaviour (susceptible vs. cell death) in N. suaveolens are known. As indicated by infectivity assays, N. suaveolens responded hypersensitively to various TMVCPs including the type strain U1. During the course of this study, more than 300 plants were inoculated, and all of them developed local lesions implying that resistance is due to homozygous single/double insert gene(s) at 99 % probability. This is supported by the fact that HR-mediated resistance is commonly determined by a single dominant plant gene . As it is possible to identify genes involved in resistance or host range only when susceptible and resistant lines can be crossed sexually and analyzed , further analysis of resistance gene is dependent on other accessions of the species that have been reported as systemic and non-necrotic hosts for TMV . The genetic background of N. suaveolens is not fully known. However, it appears to be one of the native species found in Australian continent, and a likely hybrid from a cross between N. suaveolens and N. fragrans . Therefore, this species may represent an ancient and divergent Nicotiana species that may carry some form of R-gene that is not fully compatible with tobamoviruses; although HR local lesions form, many tobamoviruses move systemically. The identification of the appropriate R gene of N. suaveolens might give information about the evolutionary mechanisms that allow the plant to recognize elicitor molecules and how viral R genes have diverged during evolution .
TMV-30B, pTMV304 and TMV-CP (-) were obtained and used with the permission from Dr. W.O. Dawson (University of Florida, USA). TMV strains were from Dr. Hildeburg Beier (Institut für Biochemie, Bayerische Julius-Maximilians- Universität, Würzburg, Germany). Seeds of Nicotiana megalosiphon were obtained from Dr. V.A. Sisson (North Carolina State University, USA). Transgenic nahG plants were obtained from Syngenta Corp. This work was supported by Academy of Finland (project numbers 53864, 54799 and 128539), Turku University Foundation and Finnish Cultural Foundation.