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Keywords:

  • cyclin;
  • MAP kinases;
  • mRNA destabilization;
  • myb;
  • transcriptional repression

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The fungal elicitors, a xylanase from Trichoderma viride and an extract from the cell wall of Phytophthora infestans, are shown to cause a rapid reduction of the mRNA levels of various cell cycle-related genes, including MAP kinase genes and cyclin genes, in cultured tobacco cells (Nicotiana tabacum cv. Xanthi, line XD6S). Pharmacological analyses suggest that the elicitor-induced decrease in B1-type cyclin (Nicta;CycB1;3) and A1-type cyclin (Nicta;CycA1;1) mRNAs may be due to transcriptional repression, and that in D3-type cyclin (Nicta;CycD3;2) mRNA due to destabilization of the mRNA molecule itself. The activity of protein kinases is required for both the activation of defence genes and the repression of cyclin genes. The transcriptional activity of the promoter of the B1-class cyclin gene decreases upon elicitor treatment. The transactivation activity of NtmybA2, a tobacco Myb transcription activator for the M phase-specific cis-acting elements in the promoter of the B-type cyclin gene, is inhibited by elicitor treatment. In addition, the mRNA levels of NtmybA2 and two other related genes, NtmybA1 and NtmybB, decrease in response to the elicitor. Finally, we discuss a negative cross-talk between signal transduction pathways for growth and defence responses, which might be important for adaptation to environmental stress by potential pathogens.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

In nature, plants are exposed to environmental changes that force them to adapt through transcriptional reprogramming via changes in the transcriptional activity of a diverse array of genes. One source of severe environmental stress is infection by a vast number of potential pathogens (Jackson & Taylor 1996). A unique and effective defence mechanism has evolved in plants to recognize pathogen invasion and to defend against such infection (Yang, Shah & Klessig 1997). It is well known that the defensive events include the increased expression of a large number of genes that encode a diverse array of proteins including metabolic enzymes, antipathogenic proteins, and signalling molecules (Kombrink & Somssich 1995).

It has also been demonstrated that elicitors induce a down-regulation of the transcript levels of several genes concomitant with the induction of a defence response in plants. A fungal elicitor was observed to induce a decrease in the mRNA level of the PvPRP1 gene, which encodes a proline-rich protein in cultured bean (Phaseolus vulgari) cells (Sheng, D’Ovidio & Mehdy 1991). A decrease in the β-tubulin mRNA level was also observed in elicitor-treated cultured soybean (Glycine max) cells (Ebel et al. 2001). In cultured parsley (Petroselinum crispum) cells, genes for histones, p34cdc2 protein kinase, and mitotic cyclin were down-regulated by treatment with a fungal elicitor (Logemann et al. 1995). Logemann et al. (1995) showed that the histone H3 mRNA level decreased around fungal infection sites in young leaves. Therefore, the down-regulation of gene expression is also associated with defence responses.

Many studies have focused on the mechanisms of positive regulation regarding the expression of defence genes in plants, which are primarily regulated at the transcriptional level. This focus has led to the identification of several important cis-acting elements and their corresponding transcription factors, which are involved in pathogen- or elicitor-activated defence gene expression (Rushton & Somssich 1998). In contrast, information about the mechanisms involved in down-regulation of gene expression during a defence response is quite limited.

It has been shown that the rapid decrease in levels of the PvPRP1 and β-tubulin mRNAs in the elicitor-treated cells is due to mRNA destabilization (Zhang et al. 1993). The molecular factors for mRNA destabilization, however, remain unknown (Ebel et al. 2001). The elicitor-induced down-regulation of genes for histones, p34cdc2 protein kinase, and mitotic cyclin in parsley cells was shown to be due to transcriptional repression by a run-on transcription assay (Logemann et al. 1995). However, the specific cis-acting elements and transcription factors involved in this down-regulation have not been reported.

We have recently been investigating the elicitor signal transduction pathway and the regulatory mechanism of defence gene expression in cultured tobacco cells (Nicotiana tabacum cv. Xanthi, line XD6S) using a cell wall extract of Phytophthora infestans and a purified xylanase from cultures of Trichoderama viride. These fungal elicitors induce various kinds of defence responses including an oxidative burst (Yano, Suzuki & Shinshi 1999), the alkalinization of the culture medium (Fukuda 1996), the expression of a subset of defence genes (Fukuda & Shinshi 1994; Suzuki, Fukuda & Shinshi 1995), and hypersensitive cell death in cultured tobacco cells (Yano et al. 1998). We have also demonstrated that a MAP kinase is involved in elicitor signalling (Suzuki et al. 1995; Suzuki, Yano & Shinshi 1999) and that a specific cis-acting element and transcription factors are involved in the elicitor-induced transcription of defence genes (Yamamoto, Suzuki & Shinshi 1999). Further investigation has led to the discovery that the transcript levels of several genes that encode MAP kinases and cyclins decrease in response to elicitor treatment. In this study, we focused on the mechanism of elicitor-induced down-regulation of the expression of tobacco cyclin genes. We showed that elicitor-induced decrease of the mRNAs of A1- and B1-type cyclin genes may be caused by decrease in transcription, whereas that of D3-type cyclin gene is caused by degradation of its own mRNA. We further analysed the mechanisms of the repression of the promoter activities of B1-type cyclin genes.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plant material and treatments

The culture of tobacco cells and the various treatments of the cells with elicitors and inhibitors has been described previously (Suzuki et al. 1995; Yano et al. 1998). Briefly, suspension cultures of tobacco cells (Nicotiana tabacum cv. Xanthi; line XD6S) were maintained at weekly intervals in a liquid medium that contained Murashige–Skoog salts (Wako Pure Chemicals Industries, Osaka, Japan), 3% sucrose, and 5 µm 2,4-dichlorophenoxyacetic acid. A suspension of XD6S cells (subcultured for 4 d) was transferred to a plastic dish that contained 2-(N-morpholino)ethanesulfonic acid (pH 5.8; final concentration, 25 m m) to stabilize the pH of the medium. The elicitor preparation (PiE) from Phytophtora infestans, a xylanase (TvX) from Trichoderma viride (Sigma, St. Louis, MO, USA), actinomycin D (Sigma), cycloheximide (Sigma), and staurosporine (Kyowa Medex, Tokyo, Japan) were added to the medium at the concentrations indicated in the Figure legends. After incubation for an appropriate period, cells were harvested by filtration with a CellStrainer (Nippon Becton Dickinson, Tokyo, Japan) and subjected to further analyses, such as measurement of fresh weight, isolation of total RNA, and LUC assay.

RNA gel blot analysis

The isolation of RNA and RNA gel-blot analysis were performed as described previously (Suzuki et al. 1995). The total RNA was extracted from tobacco cells, and aliquots of 5 µg of total RNA were denatured, fractionated by electrophoresis on a formaldehyde-agarose gel, transferred to a GeneScreen Plus membrane (NEN, Boston, MA, USA), and allowed to hybridize with 32P-labelled cDNA probes. Equal loading of total RNA samples was confirmed by visualization of rRNA that had been stained with ethidium bromide (Sigma). For use as probes, cDNA fragments for cyclin genes, including Nicta;CycA1;1 (D50735, Setiady et al. 1995), Nicta;CycB1;3 (D89635, Ito et al. 1997), and Nicta;CycD3;2 (AJ011894, Sorrell et al. 1999) were amplified from the elicitor-treated tobacco cells by reverse transcriptase-polymerase chain reaction using specific primers.

Transient assay

The construction of the effector plasmid, CaMV35S-NtmybA2, for the transient expression of NtmybA2, and the reporter plasmids Catro;CycB1;1 promoter-LUC were previously described (Ito et al. 2001).

Particle bombardments were carried out with a Biolistic PDS-1000/He Particle Delivery system (Bio-Rad, Richmond, CA, USA), as described previously (Fujimoto et al. 2000). Suspensions of XD6S cells subcultured for 4 d were used for the bombardment. LUC assays were performed with a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) using a luminescence reader (TD-20/20; Promega). The Renilla LUC gene (Promega) under the control of the CaMV35S promoter was used as an internal control. To normalize values after each assay, the ratio of LUC activity (firefly LUC/Renilla LUC) was calculated.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Elicitor-induced down-regulation of cell cycle-related gene expression

To further investigate the role of MAP kinase(s) in the elicitor signalling process, we examined mRNA levels of genes for tobacco MAP kinases Ntf3 (Wilson et al. 1993), Ntf4, Ntf6 (Wilson et al. 1995), and WIPK (Seo et al. 1995) in tobacco cells treated with either a cell wall extract of Phytophthora infestans (PiE) or a xylanase from Trichoderma viride (TvX) as fungal elicitors. The results are shown in Fig. 1a. The mRNA level of the gene for class I basic chitinase (BCHN) increased in the tobacco cells upon treatment with the fungal elicitors, which is consistent with our previous report and confirms that cells do respond to the elicitor in our experimental system (Fukuda & Shinshi 1994; Suzuki et al. 1995). Ntf4 mRNA was maintained at a constant level before elicitor treatment and was unchanged by the elicitor treatment. In control cells cultured without elicitors, a rapid and very short-term increase in the WIPK mRNA level was observed. It is possible that this induction was due to the mechanical stress associated with transferring the cell suspension to a new plastic dish for the elicitor treatment (Suzuki & Shinshi 1995; Bögre et al. 1996; Suzuki 2002). The WIPK mRNA level was also rapidly and transiently but extendedly increased after treatment with the fungal elicitors (Fig. 1a). Ntf4 is highly homologous to another tobacco MAP kinase gene, SIPK. Like Ntf4, the expression of the SIPK gene has also been observed to be constant in fungal elicitor-treated tobacco cells (Zhang, Du & Klessig 1998). In contrast, the Ntf3 and Ntf6 genes were constitutively expressed when cells were cultured without elicitors, but a marked decrease in this expression was observed after the addition of the fungal elicitors (Fig. 1). Ntf6 has been implicated in cell division in plant cells (Calderini et al. 1998). These results suggest that the fungal elicitors concomitantly induce the expression of defence-related genes, such as the chitinase gene, and reduce the expression of cell cycle-related genes.

image

Figure 1. Down-regulation of mRNA levels of genes for MAP kinases and cyclins by fungal elicitors in tobacco cultured cells. Tobacco XD6S cells were treated with water (CON) as a control, the elicitor from Phytophthora infestans (PiE) at 100 µg mL−1, and a xylanase from Trichoderma viride (TvX) at 2 µg mL−1. The total RNA was prepared and the transcript levels of genes for class I basic chitinase (BCHN), WIPK, Ntf3, Ntf4, and Ntf6 (a), and also Nicta;CycA1;1 (CycA1.1), Nicta;CycB1;3 (CycB1.3), and Nicta;CycD3;2 (CycD3.2) (b) were analysed after incubation for 0, 0.5, 1, 3, and 6 h. For the gel blot analysis, loading of equal amounts of samples was confirmed by staining the total RNA with ethidium bromide (EtBr).

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We examined the transcript levels of genes for tobacco A1-type cyclin, Nicta;CycA1;1 (Setiady et al. 1995), B1-type cyclin, Nicta;CycB1;3 (Ito et al. 1997), and the D3-type cyclin, Nicta;CycD3;2 (Sorrell et al. 1999) before and after treatment with the fungal elicitors. Substantial levels of expression of these genes have been observed in actively proliferating tobacco cells in suspension cultures. The results are shown in Fig. 1b. Since tobacco cells in the logarithmic phase of growth were used in these experiments, these cyclin genes were constantly expressed. The fungal elicitors were shown to induce a marked decrease in the mRNA levels of the cyclin genes, similar to that observed for Ntf3 and Ntf6. The mRNAs of the cyclin genes were not detectable at all 3 h after elicitor treatment. The decrease of mRNAs for cyclins suggests consequent cessation of cell division. To confirm this, we examined the effect of an elicitor on the growth of tobacco cells. As shown in Fig. 2, the growth of XD6S cells was significantly impaired in the presence of TvX. The increase in the fresh weight was arrested during the elicitor treatment concomitantly with the decrease of mRNAs for cyclins.

image

Figure 2. Effect of fungal elicitors on the growth of tobacco cultured cells. Tobacco XD6S cells were treated with water (CON) as a control and a xylanase from Trichoderma viride (TvX) at 2 µg mL−1. Fresh weight was measured at 0, 12, and 24 h after elicitation. Data are represented as the average of three independent experiments. The standard deviation is indicated by the vertical bars.

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Pharmacological analyses of elicitor-induced down-regulation of cyclin gene expression

We examined changes in mRNA levels of cyclin genes in the presence of actinomycin D, a transcription inhibitor, and compared these changes with the elicitor-induced decrease. The results are shown in Fig. 3. Nicta;CycA1;1 mRNA and Nicta;CycB1;3 mRNA levels dramatically decreased in tobacco cells treated with actinomycin D alone (Fig. 3a & b). The pattern of the decrease in the Nicta; CycB1;3 mRNA levels in this experiment was comparable to that in cells treated with the elicitor, whereas the actinomycin D-induced decrease in the Nicta;CycA1;1 mRNA level was slower than the elicitor-induced decrease and still detectable at 3 h after the addition of actinomycin D. It has been well documented that the induced accumulation of BCHN mRNA in tobacco cells treated with fungal elicitors is due to an increase in transcription (Fukuda & Shinshi 1994; Suzuki et al. 1995; Yamamoto et al. 1999). The elicitor-induced increase in the BCHN mRNA level was completely absent in the presence of actinomycin D, as shown in Fig. 4c, which indicates that the transcription of genes is effectively prevented by actinomycin D treatment in this experimental system. These results suggest that the elicitor-induced decrease in the levels of the mRNAs of Nicta;CycB1;3 was primarily due to transcriptional repression and the elicitor-induced repression is involved at least in part in the down-regulation of Nicta;CycA1;1 gene. In contrast, a decrease in the Nicta;CycD3;2 mRNA level was not observed in the cells treated with actinomycin D. This further suggests that the elicitor treatment induced a destabilization of the Nicta;CycD3;2 mRNA in tobacco cells.

image

Figure 3. Effect of actinomycin D on mRNA levels of genes for cyclins in tobacco cultured cells. Tobacco XD6S cells were treated with water (CON) as a control, a xylanase from Trichoderma viride (TvX) at 1 µg mL−1, and actinomycin D (ActD) at 100 µg mL−1. The total RNA was prepared and the transcript levels of genes for Nicta;CycA1;1 (CycA1.1, a), Nicta;CycB1;3 (CycB1.3, b), and Nicta;CycD3;2 (CycD3.2, c) were analysed after incubation for 0, 0.5, 1, 2, 3, and 6 h. For the gel blot analysis, loading of equal amounts of samples was confirmed by staining the total RNA with ethidium bromide (d, EtBr).

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image

Figure 4. Effects of actinomycin D, cycloheximide, and staurosporine on the elicitor-inducible down-regulation of mRNA levels of genes for cyclins in tobacco cultured cells. Tobacco XD6S cells were treated with water (CON) as a control and a xylanase from Trichoderma viride (TvX) at 1 µg mL−1 with or without the inhibitors staurosporine (a, STA) at 1 µm, cycloheximide (b, CHX) at 35.5 µm, or actinomycin D (c, ActD) at 100 µg mL−1. The total RNA was prepared and the transcript levels of genes for Nicta;CycA1;1 (CycA1.1), Nicta;CycB1;3 (CycB1.3), Nicta;CycD3;2 (CycD3.2), and class I basic chitinase (BCHN) were analysed after incubation for 0, 3, and 6 h. For the gel blot analysis, loading of equal amounts of samples was confirmed by staining the total RNA with ethidium bromide (EtBr).

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We further assessed the relationship of protein kinase activity, protein synthesis de novo, and RNA synthesis de novo in the elicitor-induced decay of cyclin mRNAs. Staurosporine, a serine/threonine protein kinase inhibitor, and cycloheximide, a protein synthesis inhibitor, have been shown to be effective in the cultured cells of a wide range of plant species including tobacco. Figure 3a and b confirms that the elicitor-induced expression of the BCHN gene in tobacco cells is inhibited by staurosporine and cycloheximide, as reported previously (Suzuki et al. 1995; Yamamoto et al. 1999). The elicitor-induced decrease in Nicta;CycA1;1, Nicta;CycB1;3, and Nicta;CycD3;2 mRNA levels was inhibited in the presence of staurosporine. This implies that protein kinase activity is required for the down-regulation of these transcript levels in response to elicitor treatment. Figure 4b indicates that cycloheximide prevented the elicitor-induced decrease in the mRNA levels of Nicta;CycA1;1 and Nicta;CycB1;3 but not Nicta;CycD3;2. In contrast, the Nicta;CycD3;2 mRNA level markedly decreased in cells treated with cycloheximide alone. These results suggest that de novo-synthesized and/or labile protein factors are required for the down-regulation of Nicta;CycA1;1 and Nicta;CycB1;3 genes in elicitor-treated cells and for the stability of Nicta;CycD3;2 mRNA in cells under normal culture conditions. Furthermore, actinomycin D inhibited the elicitor-induced decrease in the Nicta;CycD3;2 mRNA level, suggesting that RNA synthesis de novo is required for the down-regulation of the Nicta;CycD3;2 gene.

Transcriptional repression of the B-type cyclin gene in elicitor-treated tobacco cells

The effects of elicitor treatment on the transcriptional activity of the promoter of the B1-type cyclin gene were investigated. Both the tobacco Nicta;CycB1;3 gene and a closely related gene, Catro;CycB1;1, from Catharanthus roseus encode B1-type cyclins and are expressed specifically during the M phase of cell cycle in synchronized cultures (Ito et al. 1997). In this study, we used a chimeric gene (Fig. 5a) in which the Catro;CycB1;1 promoter is fused to a firefly luciferase (LUC) gene as a reporter (Ito et al. 1998). In synchronized transgenic BY2 cells that harboured the chimeric gene, the CycB1 promoter can direct G2/M phase-specific transcription of the LUC gene (Ito et al. 1998). We examined the effects of the elicitor treatment on transcription activity of the CycB1 gene promoter through transient assay experiments using particle bombardment. Figure 5b shows that LUC activity in cells bombarded with the CycB1 promoter-LUC plasmid was markedly reduced by approximately 84% in the presence of the fungal elicitor. This result is consistent with the elicitor-induced down-regulation of the CycB1 gene observed in the results shown in Fig. 1. Taken together, the results in Figs 1, 2 and 4 indicate that elicitor-induced down-regulation of the expression of the B-type cyclin gene is caused, at least in part, by repression of the transcription.

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Figure 5. Elicitor-induced down-regulation of transcription via the Catro;CycB1;1 promoter in tobacco cells. (a) A schematic representation of the reporter, Catro;CycB1;1 promoter-LUC (Ito et al. 2001), used in the transient assay with particle bombardment. The numbers above the grey bar indicate the distance from the Catro;CycB1;1 transcription start site. (b) Elicitor-inducible down-regulation of transcription via the Catro;CycB1;1 promoter in tobacco cells. The reporter plasmid, Catro;CycB1;1 promoter-LUC, was co-bombarded with a reference reporter plasmid, CaMV35S-Renilla LUC, into XD6S cells. After bombardment, the cells were incubated in normal culture conditions for 18 h, and were then treated with (filled column) and without (open column) TvX at 2 µg mL−1 for 6 h. All LUC activities are expressed relative to the -TvX (value set at 1). Data are represented as the average of three independent experiments. The standard deviation is indicated by the horizontal bars.

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Elicitor-induced repression of transcription by a transcription factor involved in the expression of the B-type cyclin gene

A cis-acting element, MSA (M-specific activator), has been identified as essential for G2/M phase-specific transcription in tobacco cells (Ito et al. 1998, 2001). Furthermore, three tobacco myb-like transcription factors, namely NtmybA1, NymybA2, and NtmybB, involved in the MSA-mediated transcription have been identified (Ito et al. 2001). Ito et al. (2001) have shown that NtmybA1 and NtmybA2 are transcriptional activators, whereas NtmybB is a repressor, and NtmybA2 has a predominant function in regulation of the MSA element-mediated transcription. A Catro;CYCB1;1 promoter contains four MSA elements (Ito et al. 1998; Fig. 6a. Therefore, the effect of an elicitor on the transactivation activity of NtmybA2 for transcription mediated by the CYCB1 promoter was further examined. As a control, tobacco XD6S cells were co-bombarded with a blank effector plasmid and a reporter plasmid, CYCB1 promoter-LUC. As shown in Fig. 6b, the CYCB1 promoter-mediated transcription in the bombarded cells was markedly reduced in the presence of TvX. The activity of CYCB1 promoter-mediated transcription increased approximately 2.7-fold by co-bombardment with the CaMV35S-NtmybA2 as an effector in tobacco cells without the elicitor (Fig. 6b). The NtmybA2-induced transcription markedly decreased by approximately 81% in the presence of an elicitor (Fig. 6b). This is almost equal to the level of activity of the promoter in  the  presence  of  TvX  in  tobacco  cells  bombarded  with  the blank effector plasmid. These results suggest that the elicitor treatment reduce the activity of NtmybA2 as a transactivator.

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Figure 6. Elicitor-induced down-regulation of transcription via Catro;CycB1;1 promoter in tobacco cells bombarded with the Catro;CycB1;1 promoter-LUC and CaMV35S-NtmybA2. (a) A schematic representation of the reporter plasmid, Catro;CycB1;1 promoter-LUC, and effector plasmid, CaMV35S-NtmybA2 (Ito et al. 2001), used in the transient assay with particle bombardment. The numbers above the grey bar indicate the distance from the Catro;CycB1;1 transcription start site. (b) The reporter and effector plasmids were co-bombarded with a reference reporter plasmid, CaMV35S-Renilla LUC, into the XD6S cells. After bombardment, the cells were incubated in normal culture conditions for 18 h, and then treated with (filled columns) or without (open columns) TvX at 2 µg mL−1 for 6 h. As a control, the reporter plasmid was bombarded with a blank effector plasmid, which contains no coding sequence for NtmybA2. All LUC activities are expressed relative to the data of blank plasmid/–TvX (value set at 1). Data are represented as the average of three independent experiments. The standard deviation is indicated by the horizontal bars.

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Elicitor-induced down-regulation of the expression of genes for Ntmybs in tobacco cells

The expression patterns of the genes for the three Ntmybs in tobacco cells treated with a fungal elicitor were investigated. Figure 7 shows that the mRNAs for Ntmybs were constantly detectable in untreated cells. In the cells treated with the fungal elicitor, the mRNA levels for the Ntmybs decreased. This observation was in contrast to a marked increase in the mRNA level of a chitinase gene. The levels of mRNAs for NtmybB and Nicta;CycB1;3 decreased in parallel after the addition of an elicitor. The mRNA levels for NtmybA1 and NtmybA2 also decreased, but with slower kinetics. These results suggest that the elicitor-induced transcriptional down-regulation of the B1-type cyclin gene is not primarily due to down-regulation of transcript levels of the genes for the activators, or up-regulation of transcript level of the gene for the repressor.

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Figure 7. Down-regulation of mRNA levels of genes for Ntmybs in cultured tobacco cells. Tobacco XD6S cells were treated with water (CON) as a control and a xylanase from Trichoderma viride (TvX) at 2 µg mL−1. The total RNA was prepared and the transcript levels of genes for class I basic chitinase (BCHN), Nicta;CycB1;3 (CycB1.3), NtmybA1, NtmybA2, and NtmybB were analysed after incubation for 0, 0.5, 1, 3, and 6 h. For the gel blot analysis, loading of equal amounts of samples was confirmed by staining the total RNA with ethidium bromide (EtBr).

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The results presented in this study clearly indicate that fungal elicitors induce a rapid and dramatic decrease in the transcript levels of several genes for MAP kinases and cyclins. The results presented in Fig. 4 suggest the elicitor-induced down-regulation of cyclin genes in tobacco cells requires the activity of serine/threonine protein kinases. Our previous studies also showed that serine/threonine protein kinases are involved in the diverse array of elicitor-induced defence responses in tobacco cells (Suzuki et al. 1995, 1999; Suzuki & Shinshi 1995; Yano et al. 1998). In plants, serine/threonine protein kinases, such as MAP kinase cascades, calcium-dependent protein kinases, and receptor kinases, have been implicated in elicitor signal transduction (Suzuki & Shinshi 1996; Romeis 2001; Peck 2003). These observations suggest that the cyclin genes are down-regulated concomitantly with the activation of defence responses downstream of the elicitor signal transduction.

The present results suggest that the elicitor-induced decrease in the Nicta;CycD3;2 mRNA level in tobacco cells, and likewise the PvPRP1 mRNA level in bean cells (Zhang et al. 1993), is mainly due to destabilization of the mRNA molecule, which is dependent on RNA synthesis de novo. AUUUA motifs present in the 3′-untranslated region (UTR) of mRNAs, which represent a common determinant of the stability of mRNAs that encode a variety of proto-oncoproteins, cytokines, and transcription factors in mammalian cells, are also believed to play important roles in the post-transcriptional regulation of gene expression in plants (Gutiérrez, MacIntosh & Green 1999). It has been demonstrated that the AUUUA motifs are present in the 3′-UTR of PvPRP1 mRNA (Zhang et al. 1993), but none have been found in the 3′-UTR of Nicta;CycD3;2 mRNA. Therefore, it is possible that the destabilization of mRNAs for tobacco Nicta;CycD3;2 and bean PvPRP1 is regulated via somewhat different mechanisms.

The present results suggest that the elicitor-induced transcriptional repression and destabilization of mRNA might be involved in the down-regulation of the Nicta;CycA1;1 gene in tobacco cells. It has been shown that oxidative stress down-regulates the expression of Nicta;CycA1;1 genes in tobacco cells (Reichheld et al. 1999). This suggests that elicitor-induced oxidative stress might be involved in the down-regulation of Nicta;CycA1;1 genes, but no information was available to illustrate the molecular mechanism of the transcriptional repression and the destabilization of mRNA of Nicta;CycA1;1 genes.

On the other hand, the present results suggest that the elicitor-induced decrease of mRNAs for Nicta;CycB1;3 gene in tobacco cells is primarily due to transcriptional repression. The expression of Nicta;CycB1;3 and other plant B-type cyclin genes has been shown to be G2/M phase-specific (Setiady et al. 1995; Fuerst et al. 1996; Ito et al. 1997). A recent study indicated that the MSA element in promoters of plant B-type cyclin genes, including Nicta;CycB1;3, is responsible for the G2/M phase-specific transcription (Ito et al. 1998). This indicates the MSA element-mediated transcription is crucial for the expression of the Nicta;CycB1;3 gene during the logarithmic growth phase in cultured tobacco cells. Therefore, it is reasonable to speculate that repression of MSA element-mediated transcription is involved in the elicitor-induced down-regulation of Nicta;CycB1;3. In addition, it is possible that elicitors cause changes in the balance between the nuclear concentration of the transcriptional activators and the repressor, which, in turn, causes the repression of the transcription of the Nicta;CycB1;3 gene. A possible involvement of increase in the production of a repressor protein, NtmybB, in the down-regulation of the Nicta;CycB1;3 gene can be excluded, because the transcript level of the NtmybB gene rapidly and markedly decreased concurrently with the decrease in the Nicta;CycB1;3 mRNA level after the addition of an elicitor. The transcript levels of NtmybA1 and NtmybA2 also decreased in the presence of an elicitor, but much slower in comparison with the Nicta;CycB1;3 and NtmybB mRNAs. Additionally, elicitor treatment reduced NtmybA2-induced transactivation of the promoter of B1-type cyclin gene (Fig. 6b). A recent report demonstrated that the activity of NtmybA2 is controlled post-translationally (Araki et al. 2004). Taken together, we can postulate that the elicitor-induced inactivation of NtmybA2, that is caused by post-translational modulation, might be involved in the elicitor-induced down-regulation of the Nicta;CycB1;3 gene.

The promoter of the NACK1 gene, which is expressed at similar phases in the cell cycle as B-type cyclin genes, also contains functional MSA-like elements that direct G2/M phase-specific transcription (Ito et al. 2001). NACK1 was identified as an activator of a MAP kinase kinase kinase NPK1, in tobacco cells (Nishihama et al. 2002). NACK1, NPK1 and its downstream kinases, NtMEK1/NQK1 and Ntf6/NRK1, have been implicated in the regulation of plant cytokinesis (Calderini et al. 1998, 2001; Préstamo et al. 1999; Nishihama et al. 2001, 2002; Soyano et al. 2003). We observed that NACK1 promoter-mediated transcription was also markedly reduced in elicitor-treated tobacco cells (unpublished result). This is consistent with the elicitor-induced down-regulation of the Ntf6 gene (Fig. 1a).

Although the biological significance of down-regulation of the expression of cell cycle-related genes in growing plant cells by pathogen-derived elicitors is unclear at this time, the present results suggest that a signal transduction pathway of growth cues is adversely affected by the elicitor signal transduction pathway concomitant with activation of the defence response in plants. Consistently with this, it has been reported that a bacterial peptide elicitor causes the activation of defence responses and the strong growth inhibition in Arabidopsis seedlings cultured in liquid medium (Gómez-Gómez, Felix & Boller 1999). In addition, both dwarfism and the constitutive expression of defence responses have been shown to be present in the same mutant plants of Arabidopsis, including constitutive triple response 1 (ctr1; Kieber et al. 1993), mpk4 (Petersen et al. 2000), constitutive expression of VSP1/ectopic lignification 1 (cev1/eli1; Ellis & Turner 2001; Caño-Delgado et al. 2003), suppressor of npr1–1, inducible 1 (sni1; Li et al. 1999), and constitutive expressor of PR genes (cpr) mutants (Bowling et al. 1994). Thus, it is plausible that antagonistic cross-talk between signal transduction pathways for growth and the defence response in plants might exist, and that this is probably an important environmental adaptation of plants that are always at risk of infection by a vast number of potential pathogens. This is, however, only speculative at this time. We are currently in the process of identifying factors involved in elicitor-induced growth inhibition in plants to test this theory and to understand the details of the mechanism.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

We thank Professor Yasunori Machida at Nagoya University for providing the DNA of the promoter region of the NACK1 gene. T.N. was supported by the Japan Science and Technology Corporation as a Domestic Research Fellow.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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