JXB Advance Access originally published online on March 3, 2003
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Journal of Experimental Botany, Vol. 54, No. 385, pp. 1175-1181,
April 1, 2003
© 2003 Oxford University Press
Isolation of tobacco ubiquitin-conjugating enzyme cDNA in a yeast two-hybrid system with tobacco ERF3 as bait and its characterization of specific interaction
Received 15 July 2002; Accepted 17 December 2002
1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2 Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
3 Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1 Higashi, Tsukuba 305-0046, Japan
4 Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1 Higashi, Tsukuba 305-0046, Japan
5 Present address: Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585, Japan.
6 To whom correspondence should be addressed at the Department of Plant Gene and Totipotency, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. Fax: +81 75 753 6398. E-mail: fumihiko{at}kais.kyoto-u.ac.jp
Abbreviations: EAR, ERF-associated amphiphilic repression; ERF, ethylene-responsive factor; GAL4AD, GAL4 activation domain; GAL4BD, GAL4 binding domain; LUC, luciferase; NLP, nitrilase-like protein; SD, synthetic dropout, UBC, ubiquitin-conjugating enzyme.
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Abstract |
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Tobacco ETHYLENE-RESPONSIVE FACTOR3 (ERF3) is a member of the ERF-domain transcription factors and has a transcriptional repressor activity, whereas other ERF proteins show activation activity. To understand the regulation of ERF3-repressor activity, protein(s) were screened which interact with ERF3 in a yeast two-hybrid system. A partial sequence (B8) of NtUBC2, a tobacco ubiquitin-conjugating enzyme was isolated. This B8 specifically interacted with ERF3 in the yeast two-hybrid system. Further analyses revealed that the region unique to ERF3 interacted with B8. The physiological functions of NtUBC2 and the stability of ERF3 are discussed in relation to the regulation of the repression activity of ERF3.
Key words: Degradation, ethylene responsive factor (ERF), repressor, transient assay, two-hybrid system, ubiquitin-conjugating enzyme (UBC).
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Ethylene-responsive factors (ERFs) are plant-specific transcription factors that contain a highly conserved DNA-binding domain consisting of c. 58 amino acids. The DNA-binding domain is designated as the ERF domain (Allen et al., 1998; Ohme-Takagi et al., 2000). The complete Arabidopsis genome sequence has revealed that 124 genes code proteins that possess the ERF domain (referred to as ERF proteins) (Riechmann et al., 2000). ERF proteins have been shown to play a variety of roles in developmental processes and in defence responses to environmental stimuli (Riechmann and Meyerowitz, 1998; Ohme-Takagi et al., 2000). Many ERFs from tobacco, Arabidopsis, and tomato have been shown to bind nucleotide sequences containing the GCC box, the core sequence of an ethylene-responsive element of defence genes, and to regulate the expression of GCC box-mediated transcriptions (Ohme-Takagi and Shinshi, 1995; Sato et al., 1996; Büttner and Singh, 1997; Solano et al., 1998; Fujimoto et al., 2000; Gu et al., 2000; Park et al., 2001).
ERF proteins are grouped into one of three classes based on amino acid sequence identities within the ERF domain (Fujimoto et al., 2000). Class I and class III ERFs act as activators, whereas class II ERFs act as repressors (Ohta et al., 2000; Fujimoto et al., 2000). Class II ERF repressors have a conserved motif, ERF-associated amphiphilic repression (EAR) motif, in their C-terminal region. The domain containing the EAR motif mediates the repression activity of class II ERF repressors and can function as a repression domain (Ohta et al., 2001). Class II ERF repressors down-regulate the transactivation activity of other ERFs (Fujimoto et al., 2000). Whereas the dissociation constant of the class II ERF domain to the GCC box sequence is comparable to those of the class I and class III ERF domains (D Hao and M Ohme-Takagi, unpublished results), the class II ERF repressors can suppress the activation activity of other ERF proteins when co-expressed (Fujimoto et al., 2000; Ohta et al., 2001). To understand the GCC box-mediated transcription of defence genes, it is important to clarify the molecular mechanism not only of transactivation but also of repression of class II ERF repressors.
The transcriptional regulation of genes for class II ERF repressors have been analysed. The tobacco ERF3 gene coding for a class II repressor as well as genes for activators such as ERF2 and ERF4 is transcriptionally up-regulated in response to ethylene (Ohme-Takagi and Shinshi, 1995; Kitajima et al., 2000). These ERF genes for both activators and a repressor are rapidly induced by mechanical wounding in tobacco leaves and by a fungal elicitor in cultured tobacco cells (Suzuki et al., 1998; Yamamoto et al., 1999; Nishiuchi et al., 2002). On the other hand, ERF genes are expressed in roots of tobacco plant whereas the ERF3 gene is constitutively expressed in several lines of cultured tobacco cells (Ohme-Takagi and Shinshi, 1995; Koyama et al., 2001), suggesting the developmental control of the transcription of ERF genes.
By contrast, the post-translational regulation of class II ERF repressors is poorly understood. Since activities of many transcription factors are modified post-translationally through protein–protein interactions (Schwechheimer and Bevan, 1998), it is important to isolate proteins that interact with class II ERF repressors. In this study, proteins were screened that interacted with a tobacco class II ERF repressor (ERF3) in a yeast two-hybrid system and a partial sequence (B8) of NtUBC2, a ubiquitin-conjugating enzyme was isolated. Further characterization of the isolated clone of B8 suggested the possible involvement in the regulation of repression activity of ERF3.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant materials
Cultured NII cells derived from Nicotiana tabacum cv. Samsun NN were maintained under continuous light as described previously (Takeda et al., 1990). BY-2 cells were maintained as described previously (Nagata et al., 1992; Ohme-Takagi and Shinshi, 1995).
Constructs of clones
The MATCHMAKER Two-Hybrid System (CLONTECH, CA, USA) was used to detect the physical interaction between ERF3 and its interacting protein. The entire or deleted coding sequences of ERF3 were amplified by PCR using the appropriate primers and cloned into EcoRI–SalI sites in pGBT9. Truncated versions of ERF3 were made by PCR and cloned into EcoRI–SalI sites of pGAD424. The coding sequence of Pto (Martin et al., 1993) was cloned into EcoRI–SalI sites in pAS2-1. The coding sequence of ERF2 was cloned into BamHI–EcoRI sites in pACT2. The 4xGCC-LUC reporter plasmid, in which four copies of the GCC box are fused upstream of the firefly Luciferase (LUC) gene, was obtained from M Ohta and H Shinshi (unpublished data). Mutated NtUBC2 (NtUBC2m), in which the cysteine residue at position 88 was replaced with an alanine residue, was made by a Quick Change Site-Directed Mutagenesis Kit (Stratagene, CA, USA). Effector plasmids pNtUBC2 and pNtUBC2m for in vivo transient assay in plant cells were made by fusing the coding sequence of NtUBC2 and NtUBC2m, respectively, downstream of cauliflower mosaic virus 35S promoter as described previously (Ohta et al., 2000).
Yeast two-hybrid screening
The tobacco cDNA library was constructed using mRNA from cultured NII cells 7 d after inoculation. The cDNAs were synthesized using reverse transcriptase and T4 DNA polymerase (Amersham Pharmacia, UK), and ligated into EcoRI and SalI sites in pGAD424. Approximately 1x107 colonies containing the primary cDNAs were obtained. The plasmid library in pGAD424 was transformed into the yeast strain HF7c by a lithium acetate method (Gietz et al., 1995). Approximately 1x107 transformants were plated on synthetic dropout (SD) selection medium that lacked tryptophan, leucine and histidine. Yeast transformants that appeared on the selection medium within 5 d were streaked on a nylon membrane soaked with SD medium lacking tryptophan and leucine, and then filter-lift assays were performed as described in the protocol supplied by the manufacturer. The pGAD424 plasmids isolated from both his3- and lacZ- positive yeast transformants were co-transformed with the empty pGBT9 into the HF7c yeast strain to determine the specificity of growth.
RNA blot analysis
Total RNA was isolated from cultured NII cells at different stages or leaves, stems, roots of two-month-old N. tabacum cv. Samsun NN. Aliquots of 10 µg of RNA were loaded on agarose gel, electrophoresed and blotted onto a nylon membrane. The membrane was hybridized with the full-length fragment of the NtUBC2 cDNA labelled with 32P and exposed with X-ray films.
Luciferase assay
A DNA mixture containing 400 ng of the 4xGCC-LUC reporter plasmid, 10 ng of pPTRL (35S promoter-Renilla LUC) as a reference (Fujimoto et al., 2000), various combinations of the effector plasmids and an appropriate amount of pUC19 to adjust the total amount of plasmids was delivered to tobacco BY-2 cells by particle bombardment as described previously (Ohta et al., 2000). The BY-2 cells were incubated for 6 h in the dark at 26 °C and frozen in liquid nitrogen. Proteins were extracted to quantify LUC activity using a Dual Luciferase Reporter Assay System (Promega, CA, USA). The activity of the reporter gene was normalized with the activity of the reference Renilla LUC.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Isolation of proteins that interact with ERF3
To isolate proteins that interact with ERF3, the entire ERF3 protein fused with GAL4 binding domain (GAL4BD) was first used as bait and screened in a yeast two-hybrid system. In the yeast strain HF7c, ERF3 slightly but significantly activated the transcription of his3 and lacZ reporter genes, although it had been reported that ERF3 did not activate the transcription of reporter genes (Ohta et al., 2000). The repression domain of ERF3 activated transcription of reporter genes in yeast, suggesting that the repression activity of this domain is plant specific (M Ohta and M Ohme-Takagi, unpublished results). In this two-hybrid screening, the selection medium did not contain 3-aminotriazole and leaky expression of the his3 gene might be observed. To avoid activation of the reporter genes, the ERF3 protein in which the C-terminal 29 amino acids in the repression domain were deleted (1/196ERF3) was used as bait since it was found that 1/196ERF3 did not activate the reporter genes in the yeast HF7c. Approximately 1x107 transformants, in which 1/196ERF3 and cDNAs in pGAD424 were co-transformed, were screened by growth selection on medium lacking histidine and by the activity of ß-galactosidase. A clone designated b8 was isolated as a clone that activated transcription of the two reporter genes in the presence of 1/196ERF3. The specific interaction of B8 and 1/196ERF3 was confirmed by the re-introduction of plasmid extracted into yeast HF7c (Fig. 1A).
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b8 encoded a partial sequence of ubiquitin-conjugating enzyme
Sequence analysis revealed that clone b8 encoded the partial sequence of a ubiquitin-conjugating enzyme (UBC) fused in-frame with GAL4AD. Then, two independent full-length cDNAs, NtUBC1 and NtUBC2 (each four clones), were isolated by colony hybridization using clone b8 as a probe from the tobacco cDNA library. All these clones had a 5' untranslated region and were fused with GAL4AD to produce no functional NtUBC. NtUBC1 and NtUBC2 showed 97% nucleotide sequence identity and encoded proteins with only one difference at the tenth amino acid residue (Fig. 2). The clone b8 was missing the first 27 nucleotides of the coding sequence of NtUBC2.
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UBCs function in the conjugation of ubiquitin to substrate proteins in the ubiquitin–proteasome pathway, which carries out the selective degradation of many short-lived proteins in eukaryotic cells (Hershko and Ciechanover, 1998). The conserved cysteine residue at the active site of UBCs is involved in ubiquitin thioester formation and transfer to substrate protein(s) (Haas and Siepmann, 1997). The two NtUBCs also contained the conserved cysteine residue.
In Arabidopsis, more than 30 genes are estimated to encode UBCs (Bachmair et al., 2001). The deduced amino acid sequences of NtUBC2 were similar to those of a family of UBCs that includes AtUBC1, AtUBC2 and AtUBC3 (Fig. 2). Several expressed sequence tags of rice that might encode their UBCs were also found in the database. Whereas AtUBC1 has been shown to accept ubiquitin and transfer it to a substrate protein (Sullivan and Vierstra, 1991), the high amino acid sequence similarity between NtUBCs and AtUBC1 suggested that NtUBCs have similar activity to that of AtUBC1.
Expression of the NtUBC2 gene in plant tissues
The accumulation of NtUBC2 transcripts in various cells and different plant tissues was examined. NtUBC2 transcripts were constitutively accumulated in tobacco cells during culture (Fig. 3A). In addition, NtUBC2 transcripts were found in all tissues of tobacco examined, including leaves at different stages, stems and roots, although there were fewer NtUBC2 transcripts in younger leaves (Fig. 3B). Additional experiments also showed that the NtUBC2 expression level was not modified by the ethylene treatment in 2-week-old tobacco seedlings (data not shown). These results are consistent with the observation that AtUBC1-3 genes are constitutively expressed in many tissues of Arabidopsis at various developmental stages (Thoma et al., 1996), suggesting that this family of plant UBCs has an important role in plant development and physiology.
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Characterization of specific interaction of ERF3 and NtUBC2
Since clone b8 encoded a partial sequence of NtUBC2, the region of ERF3 that is required for interaction with the NtUBC2 protein was further characterized. ERF3 has the ERF domain in the region of amino acids 26 to 82 and the repression domain with the EAR motif in amino acids191 to 225 (Ohme-Takagi and Shinshi, 1995; Ohta et al., 2001). The entire and truncated versions of ERF3 (1/225, 1/196, 1/91, 92/196, 191/225) were cloned into pGAD424 to fuse to the GAL4 activation domain (GAL4AD) and used for the determination of interaction. Since full length cDNA of NtUBC2 fused to GAL4BD did not show the interaction with ERF3 fused to GAL4AD and also the degradation of ERF3 was suggested, the b8 cDNA fused to GAL4BD in pAS2-1 was used to characterize the specificity of the interaction in the yeast two-hybrid system (Fig. 1B). Co-transformants containing both the B8 protein and 1/225ERF3, 1/196ERF3 or 92/196ERF3 activated the transcription of reporter genes, whereas 1/91ERF3 or 191/225ERF3 did not activate the transcription of reporter genes (Fig. 1B). These results clearly indicated that 92/196ERF3 was necessary and sufficient for the interaction with the B8 protein, while either the ERF domain or the repression domain of ERF3 were insufficient for this interaction.
To test the specific interaction of B8 protein with ERF3 among ERF proteins, the interaction of the B8 protein with ERF2 in the yeast two-hybrid system was measured. As shown in Fig. 1C, yeast co-transformants containing the B8 protein and ERF2 did not activate the reporter genes, whereas Pto kinase, which has been shown to interact with ERF2 (Zhou et al., 1997), activated the transcription of the reporter genes when co-transformed with ERF2 (Fig. 1C). It was confirmed that the B8 protein did not interact with ERF4, either (data not shown). These results suggested that the B8 protein specifically interacted with ERF3.
Previously, the ERF domains of several ERFs including ERF3 were found to interact with a tobacco nitrilase-like protein (NLP) (Xu et al., 1998). Because this result shows that B8 interacted with 92/196ERF3, but not the ERF domain of ERF3, the binding specificity of NtUBC2 is suggested to be different from NLP.
Effect of NtUBC2 on the repression activity of ERF3
To analyse the in vivo function of NtUBC2 on the repression activity of ERF3, transient co-transformation assays were performed in tobacco cells. A LUC-encoding reporter gene with four copies of the GCC box and effector plasmids under the control of cauliflower mosaic virus 35S promoter were co-expressed in cultured tobacco BY-2 cells (Fig. 4A). ERF4 clearly activated the reporter gene activity whereas ERF3 repressed. The repression activity of ERF3 to the activation activity of ERF4 was dose-dependent (Fig. 4B).
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, from tobacco mosaic virus was located upstream of the translation initiation site. (B) Repression activity of ERF3 in BY-2 cells. Different concentrations of the ERF3 effector (from 0 to 400 ng) were co-bombarded with 400 ng of the ERF4 effector. All LUC activities were normalized by those of the reference plasmid. (C) Effects of NtUBC2 or NtUBC2m on the repression activity of ERF3. A plasmid mixture containing 20 ng of the ERF3 effector, 400 ng of the ERF4 effector and 400 ng of either the NtUBC2 effector or the NtUBC2m effector was co-bombarded with the reporter plasmid and the reference plasmid into BY-2 cells. All LUC activities were normalized by the activity of the reference plasmid. Each value is the average of 6–12 independent measurements.To analyse the effect of the ubiquitin-conjugation activity of NtUBC2, a mutation was generated in which the cysteine residue at position 88 in the active site of NtUBC2 was replaced by an alanine residue. This mutant (NtUBC2m) was expected to disrupt the ubiquitin-conjugating activity of NtUBC2 as observed in AtUBC1 (Sullivan and Vierstra, 1993). Expression of NtUBC2 or NtUBC2m did not affect the activity of the basal reporter gene nor the activation activity of ERF4 (Fig. 4C), indicating that the expression of NtUBC2 and NtUBC2m did not affect general transcription, LUC activity or transactivation activity of ERF4 in this assay. A mixture of 20 ng of the ERF3 effector and 400 ng of the ERF4 effector was used to induce a moderate reduction of the reporter gene activity. Co-bombardment of the NtUBC2 effector with this mixture did not affect the reporter gene activity, while co-bombardment of the NtUBC2m effector significantly reduced the reporter gene activity. Statistical analysis using t-test indicated that the variance (difference) was significant more than 95% probability. Since NtUBC2m did not affect the basal reporter gene activity or transactivation activity of ERF4, this result suggested that NtUBC2m increased the repression activity of ERF3.
It is speculated that ERF3 is a substrate of NtUBC2, since the repression activity of ERF3 was enhanced by the dominant-negative inhibition of ubiquitin–conjugation acitivity of NtUBC2 in transient gene expression assays. Several reports have shown that expression of catalytic-site-mutants of UBCs inhibits the ubiquitination and degradation of substrates (Lo and Massague, 1999; Pati et al., 1999). The ubiquitin-conjugation activity of endogenous NtUBC2 is likely to be involved in the regulation of repression activity of ERF3. The stability of ERF3 might be increased by the expression of NtUBC2m. In contrast to NtUBC2m, NtUBC2 did not have any discernible effect on the repression activity of ERF3. It is possible that the endogenous activity of NtUBC2 might be sufficient for modulation of the repression activity of ERF3. Alternatively, expression of other components in the ubiquitin–proteasome pathway such as a ubiquitin ligase might be required for the activity of NtUBC2.
It has been shown that NtUBC2 interacted with ERF3 but not with ERF2 or ERF4. This suggests that the mechanism of regulation of the repression activity of ERF3 is distinct from that of the activation activity of ERF activators. Since ERF repressors can suppress transactivation activity of ERF activators (Fig. 4B; Fujimoto et al., 2000), down-regulation of the repression activity of ERF3 should be operating for the induction of the GCC box-mediated transcription of defence genes. Moreover, the observation that external stresses such as ethylene and a fungal elicitor induce expression of ERF genes for both activators and a repressor (Ohme-Takagi and Shinshi, 1995; Yamamoto et al., 1999) suggests that the repression activity might be down-regulated at a post-transcriptional level, such as the control of the protein stability. Thus, the interaction between ERF3 and NtUBC2 is likely to be a critical step for the down-regulation of the repression activity of ERF3.
Proteolysis by the ubiquitin–proteasome pathway has been implicated in many aspects of development, signalling in hormone responses, light perception and circadian rhythm in plants (Gray et al., 2001; Callis and Vierstra, 2000; Bachmair et al., 2001; Schwechheimer and Deng, 2001; Hellmann and Estelle, 2002). The ubiquitin–proteasome pathway has been shown to be involved in signal transduction in response to auxin, cytokinin, brassinosteroid and jasmonic acid, whereas the involvement of this proteolytic pathway in ethylene signalling is still unknown (Gray et al., 2001; Schwechheimer et al., 2001; Xie et al., 2002; Smalle et al., 2002; He et al., 2002; Xu et al., 2002). Further studies of protein stability of ERF3 should help to clarify the fine-tuning mechanisms of transcription based on ERF activators and ERF repressors.
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Acknowledgements |
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We thank Dr Gregory Martin for a kind gift of Pto cDNA. We are grateful to Drs Kaoru Suzuki and Masaru Ohta for fruitful discussions. This research was supported in part by Grant-in-aid for Research for the Future Program Grant JSPS-RFTF00L01606 from the Japan Society for Promotion of Science (to FS) and a JSPS Fellowship for Japanese Junior Scientists (to TK). The accession numbers are NtUBC1 (AB026055), NtUBC2 (AB026056).
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 A. Andriankaja, A. Boisson-Dernier, L. Frances, L. Sauviac, A. Jauneau, D. G. Barker, and F. de Carvalho-Niebel AP2-ERF Transcription Factors Mediate Nod Factor Dependent Mt ENOD11 Activation in Root Hairs via a Novel cis-Regulatory Motif PLANT CELL, September 1, 2007; 19(9): 2866 - 2885. [Abstract] [Full Text] [PDF]
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 K. C. McGrath, B. Dombrecht, J. M. Manners, P. M. Schenk, C. I. Edgar, D. J. Maclean, W.-R. Scheible, M. K. Udvardi, and K. Kazan Repressor- and Activator-Type Ethylene Response Factors Functioning in Jasmonate Signaling and Disease Resistance Identified via a Genome-Wide Screen of Arabidopsis Transcription Factor Gene Expression Plant Physiology, October 1, 2005; 139(2): 949 - 959. [Abstract] [Full Text] [PDF]
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T. Ogawa, L. Pan, M. Kawai-Yamada, L.-H. Yu, S. Yamamura, T. Koyama, S. Kitajima, M. Ohme-Takagi, F. Sato, and H. Uchimiya Functional Analysis of Arabidopsis Ethylene-Responsive Element Binding Protein Conferring Resistance to Bax and Abiotic Stress-Induced Plant Cell Death Plant Physiology, July 1, 2005; 138(3): 1436 - 1445. [Abstract] [Full Text] [PDF]
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