Dysfunctionality of a tobacco mosaic virus movement protein mutant mimicking threonine 104 phosphorylation

E. M. Karger1, O. Yu. Frolova1, N. V. Fedorova1, L. A. Baratova1, T. V. Ovchinnikova2, P. Susi3, K. Makinen4, L. Ronnstrand5,{dagger}, Yu. L. Dorokhov1 and J. G. Atabekov1

1 Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Vorobiovy Gory Moscow 119899, Russia
2 M. M. Shemyakin and Yu. A. Ovchinnikov Institut of Bioorganic Chemistry, Moscow, Russia
3 Joint Biotechnology Laboratory, Biocity, Turku, Finland
4 University of Helsinki, Institute of Biotechnology, Biocenter, Helsinki, Finland
5 Ludwig Institute of Cancer Research, Biomedical Center, Uppsala, Sweden

Correspondence
Joseph Atabekov
atabekov{at}genebee.msu.su


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Replication of tobacco mosaic virus (TMV) is connected with endoplasmic reticulum (ER)-associated membranes at early stages of infection. This study reports that TMV movement protein (MP)-specific protein kinases (PKs) associated with the ER of tobacco were capable of phosphorylating Thr104 in TMV MP. The MP-specific PKs with apparent molecular masses of about 45–50 kDa and 38 kDa were revealed by gel PK assays. Two types of mutations were introduced in TMV MP gene of wild-type TMV U1 genome to substitute Thr104 by neutral Ala or by negatively charged Asp. Mutation of Thr104 to Ala did not affect the size of necrotic lesions induced by the mutant virus in Nicotiana tabacum Xanthi nc. plants. Conversely, mutation of Thr to Asp mimicking Thr104 phosphorylation strongly inhibited cell-to-cell movement. The possible role of Thr104 phosphorylation in TMV MP function is discussed.

Published ahead of print on 20 December 2002 as DOI 10.1099/vir.0.18972-0.

Present address: Division of Experimental Clinical Chemistry, Lund University, Malmo University Hospital, Malmo, Sweden.


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Cell-to-cell movement of tobacco mosaic virus (TMV) is mediated by a 30 kDa movement protein (MP) encoded by TMV RNA (reviewed by Carrington et al., 1996; Lazarowitz & Beachy, 1999; Tzfira et al., 2000). It has been shown that P30 accumulates in plasmodesmata of TMV-infected and MP-transgenic plants (Tomenius et al., 1987; Atkins et al., 1991; Oparka et al., 1997; Heinlein et al., 1998) to increase their permeability (Wolf et al., 1991; Ding et al., 1992). The TMV MP co-aligns with microtubules (McLean et al., 1995; Heinlein et al., 1995) and is tightly associated with ER-derived membranes of infected cells (Reichel & Beachy, 1998; Heinlein et al., 1998). Furthermore, it has been reported that ER-enriched fractions from infected tobacco leaves contain TMV MP, RNA and replicase, implying that virus replication and protein synthesis take place in this compartment (Mas & Beachy, 1999). Importantly, it is known that P30 accumulates in the cell wall (CW) fraction of transgenic plants as a phosphoprotein (Citovsky et al., 1993; Waigmann et al., 2000) and that the CW-associated protein kinase(s) (PKs) can use MP as substrate. C-proximal residues Ser258, Thr261 and Ser265 have been identified as phosphorylation sites in vitro (Citovsky et al., 1993) and in vivo (Waigmann et al., 2000). TMV encoding a mutant MP mimicking phosphorylation of these sites by negatively charged Asp substitution is unable to move from cell to cell in Nicotiana tabacum plants (Waigmann et al., 2000). It has been proposed that C-terminal phosphorylation of TMV MP abolishes its ability to promote virus spread (Waigmann et al., 2000).

Watanabe et al. (1992) reported that C-terminal residues 234–261 are required for TMV MP phosphorylation in tobacco protoplasts, although it is clear that in TMV-infected protoplasts MP can be phosphorylated at multiple internal phosphorylation sites (Haley et al., 1995). In particular, two distinct domains (residues 61–114 and 212–231) can be substrates for PK(s) other than the CW-associated PK(s) mentioned above. Similarly, it has been shown that Ser37 and Ser238 of tomato mosaic tobamovirus (ToMV) MP can be phosphorylated in protoplasts. The presence of Ser at position 37 or phosphorylation of Ser37 is important for ToMV MP functionality (Kawakami et al., 1999). Thus, the results from different groups of workers imply that TMV MP is phosphorylated in vivo, although it is hard to say in what cellular compartments MP phosphorylation occurs. It is important to emphasize that only the C-proximal sites of CW-associated TMV MP are found phosphorylated in planta (Waigmann et al., 2000), while multiple internal sites of MP could be phosphorylated in infected protoplasts (Haley et al., 1995; Kawakami et al., 1999). Despite the apparent contradiction, the various results are not necessarily incompatible. Thus, TMV MP might be transiently phosphorylated when subjected to processes of phosphorylation/dephosphorylation at its internal sites by cytoplasmic PK(s), whereas only the C-proximal sites are selectively phosphorylated by CW-associated PKs.

The TMV genome is accepted widely to be translocated from cell to cell as an MP–RNA complex. Moreover, it has been reported that TMV MP is an efficient repressor of in vitro translation and phosphorylation of MP prevents its translation-repressing ability (Karpova et al., 1999). Possible roles of viral MP phosphorylation in regulation of TMV genome expression have been discussed recently by Lee & Lucas (2001).

Microsomal fractions from leaves of N. tabacum var. Samsun were isolated by sucrose gradient centrifugation, as described by Mas & Beachy (1999), and analysed for PK activity using preparations of bacterially expressed TMV U1 (His)6-MP as a substrate for labelling in the presence of [{gamma}-32P]ATP. Purification of (His)6-MP was carried out as described by Karpova et al. (1997). The level of MP phosphorylation activity varied along the sucrose gradient with maximums in fractions 2–4 and 12–15 (Fig. 1a). The presence of endoplasmic reticulum (ER) lumenal-binding protein (BiP), an ER membrane resident protein (Reichel & Beachy, 1998), was revealed in fractions 2 and 11–13 by Western blotting (Fig. 1b). These data indicate that MP-specific PK activity was at a maximum in ER-containing fractions of the sucrose density gradient (Fig. 1a, b). However, there was no BiP in fractions 4 and 15 that phosphorylate the MP. Thus, the PK(s) that phosphorylate the MP may not reside exclusively in the ER.



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Fig. 1. Characterization of MP-specific ER-associated PK activity. (a) PAGE of TMV MP phosphorylated in vitro by ER-enriched sucrose gradient fractions. Identification of the MP band was performed in a series of separate Western blot assays. (b) Western blot analysis of BiP in the same fractions. Lane numbers correspond to the number of the sucrose gradient fraction. (c) Electrophoresis of TMV MP phosphorylated in vitro by a sucrose gradient fraction with high PK activity in the presence of divalent cations. Cation concentrations (mM) are indicated over the lanes. (d, e) Gel PK assays with recombinant TMV MP (d) and MBP (e) as substrates. Lane numbers correspond to sucrose density gradient fractions. Positions of TMV MP (a, c), BiP (b) and standard marker proteins (in kDa) (d) are indicated. The closed arrowhead indicates the position of kinases larger than 45 kDa; the open arrowhead denotes a minor component of 38 kDa.

 
The influence of different divalent metal cations on the MP-specific ER-associated PK activity was examined in a series of experiments. Mg2+ and particularly, Mn2+ were found to stimulate MP phosphorylation, whereas no stimulation was detected in the presence of Ca2+ (Fig. 1c). There was no PK activity when no metal cation was added to the reaction. The number and molecular masses of the MP-specific PKs in ER-associated fractions were determined by gel PK assays (Zhang & Klessig, 1997). The recombinant TMV MP and myelin basic protein (MBP), a universal substrate for mitogene-activated PKs, were compared using the assay. MP or MBP were co-polymerized with acrylamide and 20 µl of each fraction was loaded on a gel. Two or three major bands, with a molecular mass somewhat higher than 45 kDa, were revealed by this approach in ER-associated fractions when TMV MP (Fig. 1d) or MBP (Fig. 1e) was used as a substrate. It is possible that the multiple bands revealed in the ER-containing fractions by gel PK assay represent different isoforms or degradation products of MP/MBP-specific PK(s). The similarity of molecular masses (45–50 kDa) of the ER-associated MP-specific PKs described above and of mitogen-activated protein kinases Ntf4 and Ntf6 (Wilson et al., 1995) might reflect a relationship. Remarkably, the activity of PKs described in this study and that of Ntf4 and Ntf6 PKs was stimulated by Mn2+ and Mg2+. A lower molecular mass minor component (molecular mass of 38 kDa) was revealed as a minor band in the gel PK assays (Fig. 1d, open arrowhead). Matsushita et al. (2000) report that the cytoplasmic 38 kDa plant casein kinase II (CKII) is capable of phosphorylating ToMV MP and it is possible that the minor 38 kDa component detected by gel PK assay (Fig. 1d) represents CKII. Radioactive bands were not observed when the control samples (no exogenous protein added as a substrate) were analysed.

To localize the sites phosphorylated by the ER-associated PK activity in TMV MP, (His)6-MP was phosphorylated in vitro in the presence of a microsomal fraction, [{gamma}-32P]ATP and 1 mM MnCl2. After additional purification of phosphorylated protein on Ni–NTA resin in the presence of 6 M guanidium/HCl, pH 8·0 (Qiagen), according to the manufacturer's protocol, and immobilization on thiopropyl–Sepharose 6B (Sigma), the MP was digested by sequence-grade trypsin (Sigma) and the resulting peptides were analysed by two different approaches. First, two-dimensional peptide mapping was applied to separate the phosphopeptides (Fig. 2a). Analyses were performed on HTLE-7002 equipment in accordance with the manufacturer's protocol (CBS Scientific). Several 32P-labelled tryptic peptides could be seen in the phosphopeptide map (Fig. 2a), which is consistent with TMV MP being phosphorylated by ER-associated PK at multiple sites. The five most prominent spots (Fig. 2a, numbers 1–5), corresponding presumably to major 32P-labelled tryptic peptides, were subjected to phospho-amino acid analysis; it was demonstrated that 32P was incorporated into spots corresponding to phosphoserine in peptides 2–5 and to phosphothreonine in peptide 1 (data not shown). Second, the 32P-labelled phosphopeptides were separated by HPLC and isolated in sufficient amounts for partial amino acid sequencing (first five amino acids were analysed in each peptide). In the present work, we have focused on characterization of peptide 1, which was found to contain Thr104 in the N-terminal sequence ADEAT. Indirect evidence for the importance of Thr104 in TMV MP activity was provided by analyses of functional reversions of Thr104 dysfunctional mutants (Deom & He, 1997; Boyko et al., 2002). It is also noteworthy that Thr104 is conserved in MPs encoded by different ToMV (Koonin et al., 1991).



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Fig. 2. Two-dimensional mapping of 32P-labelled tryptic peptides of wild-type and mutant TMV MPs phosphorylated by ER-associated PK activity. (a) Wild-type MP, (b) T104A and (c) T104D mutant MPs. The five most prominent spots are indicated by numbers and the remaining minor spots by letters. The directions of electrophoresis and chromatography are indicated by arrows at the bottom left-hand corner.

 
To study the importance of Thr104 for function, point mutations were introduced into the recombinant (His)6-MP gene to replace Thr104 in bacterially expressed MPs with (i) alanine, which prevents phosphorylation, or (ii) aspartate, which is believed to mimic protein phosphorylation (Waigmann et al., 2000). The two mutant MP forms obtained were designated as T104A and T104D, respectively.

To examine the phosphorylation patterns of mutated MPs, the preparations of bacterially expressed recombinant T104A and T104D proteins were phosphorylated as described above and analysed by two-dimensional peptide mapping. Fig. 2(b, c) show that substitution of Thr104 by either of the amino acids Ala or Asp led to the disappearance of one major and one minor spot (Fig. 2a, labelled 1 and A) from the phosphopeptide map. The major spots, 2–5 (Fig. 2), were still present in the phosphopeptide maps of the mutant MPs. These results indicate that (i) Thr104 can be phosphorylated in vitro by ER-associated PK(s) in wild-type MP and (ii) the level of phosphorylation of phosphopeptides 2–5 was not decreased by replacement of Thr104 with either of the amino acids. It is noteworthy that two minor spots (Fig. 2a, b, labelled B and C) observed in phosphopeptide maps of wild-type and T104A MP were missing from phosphorylated T104D MP (Fig. 2c). One can speculate that substitution of Thr104 by Asp may change the MP conformation so that these two sites are not exposed to phosphorylation. Finally, the ER-associated PKs responsible for T104A, T104D and wild-type MP phosphorylation were examined by gel PK assay. The number and apparent molecular masses of PKs revealed were similar in the experiments when wild-type and mutant MPs were used as substrate (data not shown). Therefore, no particular ER-associated PK was responsible for the Thr104 phosphorylation only.

To elucidate the functional importance of Thr104 for TMV cell-to-cell movement, mutations were introduced into the MP gene of a full-length TMV U1 cDNA copy to substitute Thr104 by Ala or Asp in modified MP. The mutant viruses referred to as TMV T104A and TMV T104D, respectively, were compared by inoculation of indicator plants reacting to TMV infection by production of local lesions (N. tabacum cv. Xanthi nc.) or systemic symptoms (N. tabacum var. Samsun, N. benthamiana). Opposite halves of the same leaf were inoculated and mean values for at least 10 inoculated leaves were compared. The specific infectivity levels (number of the local lesions produced by 1·5 µg RNA on Xanthi nc. leaves) of wild-type and T104A transcripts were very similar, as was the size of lesions produced by T104A and wild-type RNA (Fig. 3a). In contrast, the specific infectivity of T104D RNA transcripts dramatically decreased (13±5 and 86±17 lesions per half-leaf were induced by T104D and wild-type TMV, respectively). It should be emphasized that only tiny local lesions were produced by T104D mutant (Fig. 3a), suggesting that the Thr to Asp substitution at position 104 strongly inhibited virus cell-to-cell movement. However, our results do not rule out that the MP produced by mutant T104D is less stable than wild-type and T104A MPs.



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Fig. 3. Dysfunctionality of TMV MP caused by substitution of Thr104 by Asp (T104D TMV) is complemented in MP-transgenic plants. Development of local lesions induced by wild-type, T104D and T104A TMV MP mutants on leaves of (a) N. tabacum cv. Xanthi nc. and (b) MP-transgenic N. tabacum cv. Xanthi nc., line 2005, plants. Mean values (with SE bars) of local lesions diameter (mm) calculated for not less than 50 local lesions are presented. (c) Western blot analysis of TMV coat protein from the upper leaves of N. tabacum var. Samsun plants infected with wild-type TMV and MP mutants T104D and T104A. Days post-inoculation (DPI) are indicated above the lanes.

 
It is important to note that the difference in development of local lesions induced by the wild-type TMV and T104D mutant was abolished when Xanthi nc. line 2005 plants transgenic for TMV MP gene were inoculated (Fig. 3b). Therefore, the movement deficiency of T104D MP could be complemented in trans by MP produced in transgenic plants. In addition, wild-type and T104A TMV induced a severe mosaic on N. tabacum var. Samsun, whereas TMV T104D mutant caused only a mild mosaic on tobacco plants. Fig. 3(c) shows that accumulation of TMV T104D in upper systemically infected leaves of N. tabacum var. Samsun plants was clearly delayed, whereas the time-course of accumulation of TMV T104A and wild-type TMV in upper leaves was similar. In order to test the stability of the T104A and T104D mutations, the progeny of the mutant viruses was isolated from N. benthamiana plants. No reversions were detected by sequencing cDNA of T104A and T104D MP genes obtained by RT-PCR. No symptom differences could be detected on N. tabacum var. Samsun and Xanthi nc. plants inoculated with primary RNA transcripts or with the progeny of mutant viruses.

In conclusion, our in vivo experiments have shown that: (i) replacement of Thr104 in TMV MP with neutral Ala did not cause significant changes in cell-to-cell movement of TMV, indicating that phosphorylation of Thr104 was not essential for MP functions; (ii) substitution of Thr104 by a negatively charged Asp residue led to a strong inhibition of the local lesion development in Xanthi nc. tobacco. This inhibition could be eliminated in Xanthi nc. plants transgenic for MP gene. Presuming that this substitution functionally mimics phosphorylation, we suggest that Thr104 phosphorylation renders TMV MP dysfunctional. If this is the case, it seems logical to hypothesize that Thr104 phosphorylation in vivo represents a defence mechanism that protects the plant from virus infections. It should be mentioned that inactivation of the MP by the Asp104 mutation may not be directly due to mimicry of phosphorylation but due to the change of the MP conformation. It is evident that our data do not provide direct evidence that MP is in fact phosphorylated at Thr104 during infection. Alternatively, it may be phosphorylated only transiently in vivo. Experiments on examination of in vivo Thr104 phosphorylation are in progress.


   ACKNOWLEDGEMENTS
 
The authors thank R. N. Beachy for generously providing the seeds of Xanthi nc. line 2005 plants, K. Lehto for providing viral full-length cDNA copy TMV 304 and V. K. Novikov for purified virus preparations. This work was supported by Fogarty International Collaboration Awards (FIRCA, grant PAR-99-008). E. K. was supported in part by The Centre for International Mobility (grant no. HAO214). P. S. was supported by Academy of Finland (grant nos 48719 and 53864).


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Atkins, D., Hull, R., Wells, B., Roberts, K., Moore, P. & Beachy, R. N. (1991). The tobacco mosaic virus 30K movement protein in transgenic tobacco plants is localized to plasmodesmata. J Gen Virol 72, 209–211.[Abstract]

Boyko, V., Ashby, J. A., Suslova, E., Ferralli, J., Sterthaus, O., Deom, C. M. & Heinlein, M. (2002). Intramolecular complementing mutations in tobacco mosaic virus movement protein confirm a role for microtubule association in viral RNA transport. J Virol 76, 3974–3980.[Abstract/Free Full Text]

Carrington, J. C., Kasschau, K. D., Mahajan, S. K. & Schaad, M. C. (1996). Cell-to-cell and long-distance transport of viruses in plants. Plant Cell 8, 1669–1681.[Free Full Text]

Citovsky, V., McLean, B. G., Zupan, J. R. & Zambryski, P. (1993). Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall-associated protein kinase. Genes Dev 7, 904–910.[Abstract]

Deom, C. M. & He, X. Z. (1997). Second-site reversion of a dysfunctional mutation in a conserved region of the tobacco mosaic tobamovirus movement protein. Virology 232, 13–18.[CrossRef][Medline]

Ding, B., Haudenshield, J. S., Hull, R. J., Wolf, S., Beachy, R. N. & Lucas, W. J. (1992). Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants. Plant Cell 4, 915–928.[Abstract/Free Full Text]

Haley, A., Hunter, T., Kiberstis, P. & Zimmern, D. (1995). Multiple serine phosphorylation sites on the 30 kDa TMV cell-to-cell movement protein synthesized in tobacco protoplasts. Plant J 8, 715–724.[CrossRef][Medline]

Heinlein, M., Epel, B. L., Padgett, H. S. & Beachy, R. N. (1995). Interaction of tobamovirus movement proteins with the plant cytoskeleton. Science 270, 1983–1985.[Abstract]

Heinlein, M., Padgett, H. S., Gens, J. S., Pickard, B. G., Casper, S. J., Epel, B. L. & Beachy, R. N. (1998). Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection. Plant Cell 10, 1107–1120.[Abstract/Free Full Text]

Karpova, O. V., Ivanov, K. I., Rodionova, N. P., Dorokhov, Yu. L. & Atabekov, J. G. (1997). Nontranslatability and dissimilar behavior in plants and protoplasts of viral RNA and movement protein complexes formed in vitro. Virology 230, 11–21.[CrossRef][Medline]

Karpova, O. V., Rodionova, N. P., Ivanov, K. I., Kozlovsky, S. V., Dorokhov, Y. L. & Atabekov, J. G. (1999). Phosphorylation of tobacco mosaic virus movement protein abolishes its translation repressing ability. Virology 261, 20–24.[CrossRef][Medline]

Kawakami, S., Padgett, H. S., Hosokawa, D., Okada, Y., Beachy, R. N. & Watanabe, Y. (1999). Phosphorylation and/or presence of serine 37 in the movement protein of tomato mosaic tobamovirus is essential for intracellular localization and stability in vivo. J Virol 73, 6831–6840.[Abstract/Free Full Text]

Koonin, E. V., Mushegian, A. R., Ryabov, E. V. & Dolja, V. V. (1991). Diverse groups of plant RNA and DNA viruses share related movement proteins that may possess chaperone-like activity. J Gen Virol 72, 2895–2903.[Abstract]

Lazarowitz, S. G. & Beachy, R. N. (1999). Viral movement proteins as probes for intracellular and intercellular trafficking in plants. Plant Cell 11, 535–548.[Free Full Text]

Lee, J. Y. & Lucas, W. J. (2001). Phosphorylation of viral movement proteins: regulation of cell-to-cell trafficking. Trends Microbiol 9, 5–8.[CrossRef][Medline]

Mas, P. & Beachy, R. N. (1999). Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA. J Cell Biol 147, 945–958.[Abstract/Free Full Text]

Matsushita, Y., Hanazawa, K., Yoshioka, K., Oguchi, T., Kawakami, S., Watanabe, Y., Nishiguchi, M. & Nyunoya, H. (2000). In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase. J Gen Virol 81, 2095–2102.[Abstract/Free Full Text]

McLean, B. G., Zupan, J. & Zambryski, P. C. (1995). Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells. Plant Cell 7, 2101–2114.[Abstract/Free Full Text]

Oparka, K. J., Prior, D. A., Santa Cruz, S., Padgett, H. S. & Beachy, R. N. (1997). Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV). Plant J 12, 781–789.[CrossRef][Medline]

Reichel, C. & Beachy, R. N. (1998). Tobacco mosaic virus infection induces severe morphological changes of the endoplasmic reticulum. Proc Natl Acad Sci U S A 95, 11169–11174.[Abstract/Free Full Text]

Tomenius, K., Clapham, D. & Meshi, T. (1987). Localization by immunogold cytochemistry of the virus-coded 30 K protein I plasmodesmata of leaves infected with tobacco mosaic virus. Virology 160, 363–371.

Tzfira, T., Rhee, Y., Chen, M. H., Kunik, T. & Citovsky, V. (2000). Nucleic acid transport in plant–microbe interactions: the molecules that walk through the walls. Annu Rev Microbiol 54, 187–219.[CrossRef][Medline]

Waigmann, E., Chen, M. H., Bachmaier, R., Ghoshroy, S. & Citovsky, V. (2000). Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein. EMBO J 19, 4875–4884.[Abstract/Free Full Text]

Watanabe, Y., Ogawa, T. & Okada, Y. (1992). In vivo phosphorylation of the 30-Kda protein of tobacco mosaic virus. FEBS Lett 313, 181–184.[CrossRef][Medline]

Wilson, C., Anglmayer, R., Vicente, O. & Heberle-Bors, E. (1995). Molecular cloning, functional expression in Escherichia coli, and characterization of multiple mitogen-activated-protein kinases from tobacco. Eur J Biochem 233, 249–257.[Abstract]

Wolf, S., Deom, C. M., Beachy, R. & Lucas, W. J. (1991). Plasmodesmatal function is probed using transgenic tobacco plants that express a virus movement protein. Plant Cell 3, 593–604.[Abstract/Free Full Text]

Zhang, S. & Klessig, D. F. (1997). Salicylic acid activates a 48-kD MAP kinase in tobacco. Plant Cell 9, 809–824.[Abstract/Free Full Text]

Received 12 November 2002; accepted 10 December 2002.