Abstract
WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of Os-WRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.
Similar content being viewed by others
References
Eulgem, T., Rushton, P. J., Robatzek, S. et al., The WRKY superfamily of plant transcription factors, Trends Plant Sci., 2000, 5: 199–206.
Wu, K. L., Guo, Z. J., Wang, H. H. et al., The WRKY family of transcription factors in rice and Arabidopsis and their origins, DNA Research, 2005, 12:9–26.
Dong, J. X., Chen, C. H., Chen, Z. X., Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response, Plant Mol. Biol., 2003, 51: 21–37.
Guo, Z. J., Kan, Y. C., Chen, X. J. et al., Characterization of a rice WRKY gene whose expression is induced upon pathogen attack and mechanical wounding, Acta. Bot. Sin., 2004, 46: 955–964.
Johnson, C. S., Kolevski, B., Smyth, D. R., TRANSPANENT TESTA GLABRA2, a trichome and seed coat development gene Arabidopsis, encodes a WRKY transcription factor, Plant Cell, 2002, 14: 1359–1375.
Zhang, Z. L., Xie, Z., Zou, X. L. et al., A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells, Plant Physiol., 2004, 134: 1500–1513.
Ishiguro, S., Nakamura, K., Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5′ upstream regions of genes coding for sporamin and α-amylase from sweet potato, Mol. Gen. Genet., 1994, 244: 563 -571.
Sun, C. X., Palmqvist, S., Olsson, H. et al., A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar-responsive elements of the isol promoter, Plant Cell, 2004, 15: 2076–2092.
Xu, Y. H., Wang, J. W., Wang, S. et al., Characterization of GaWRKYl, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-d-cadinene synthase-A, Plant Physiol., 2004, 135: 507–515.
Rushton, P. J., Torres, J. T., Parniske, M. et al., Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes, EMBO J., 1996, 15: 5690–5700.
Yang, P., Wang, Z., Fan, B. et al., A pathogen- and salicylic acidinduced WRKY DNA-binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter, Plant J., 1999, 18: 141–149.
Hara, K., Yagi, M., Kusano, T. et al., Rapid systemic accumulation of transcripts encoding a tobacco WRKY transcription factor upon wounding, Mol. Gen. Genet, 2000, 263: 30–37.
Cormack, R. S., Eulgem, T., Rushton, P. J. et al., Leucine zipper-containing WRKY proteins widen the spectrum of immediate early elicitor-induced WRKY transcription factors in parsley, Biochim. Biophys. Acta., 2002, 1576: 92–100.
Wu, J., Kurata, N., Tanoue, H. et al., Physical mapping of duplicated genomic regions of two chromosome ends in rice, Genet., 1998, 150: 1595–1603.
Thompson, J. D., Higgins, D. G., Gibson, T. J., CLUSTALW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice, Nucl. Acids Res., 1994, 22: 4673–4680.
Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, 227: 680–685.
Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed, New York: Cold Spring Harbor Laboratory Press, 1989.
Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein—dye binding, Anal. Biochem., 1976, 72: 248–254.
Zeng, W. Y., Zhang, X. C., Tu, X. M. et al., Expression and purification of the DNA-binding domain of the human transcription factor E2F1, Protein Exp. Purif, 2001, 21: 99–104.
Zhan, J. B., Chen, Y. D., Wang, K. Y. et al., Expression of ricin A chain and ricin A chain-KDEL in Escherichia coli, Protein Exp. Purif, 2004, 34: 197–201.
Makrides, S. C., Strategies for achieving high-level expression of genes in Escherichia coli, Microbiol. Rev., 1996, 60: 512–538.
Haren, L., Normand, C., Polard, P. et al., IS911 Transposition is regulated by protein-protein interactions via a leucine zipper motif, J. Mol. Biol., 2000, 296: 757–768.
Glover, M. J. N., Harrison, S. C., Crystal structure of the heterodimeric transcription factor c-Fos-c-Jun bound to DNA, Nature, 1995, 373: 257–261.
Eulgem, T., Rushton, P. J., Schmelzer, E. et al., Early nuclear events in plant defense: Rapid gene activation by WRKY transcription factors, EMBO J., 1999, 18: 4689–4699.
Du, L., Chen, Z., Identification of genes encoding receptor-like protein kinases as possible targets of pathogen and salicylic acid-induced WRKY DNA-binding proteins in Arabidopsis, Plant J., 2000, 24: 837–847.
Maeo, K., Hayashi, S., Kojima-Suzuki, H. et al., Role of conserved residues of the WRKY domain in the DNA-binding activity of tobacco WRKY family proteins, Biosci. Biotechnol. Biochem., 2001, 65: 2428–2436.
Author information
Authors and Affiliations
Corresponding author
About this article
Cite this article
Haihua, W., Zhongna, H., Ke, X. et al. Leucine zipper like structure in rice WRKY89 enhances its affinity for binding with W box elements. Chin.Sci.Bull. 50, 980–989 (2005). https://doi.org/10.1360/982005-5
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1360/982005-5