Functional characterization of a defense-related class-III chitinase promoter from Lupinus albus, active in legume and monocot tissues
A class-III chitinase promoter was isolated from Lupinus albus. The region 5′ to the coding sequence of the IF3 gene was amplified by gene walking and sequenced. The proximal 2.0 kb sequence contains a predicted promoter site, including a TATA box, near the ATG start site. To test for minimal sequences needed for promoter activity, the region was restricted into fragments of 1.81, 1.51 and 1.13 kb and cloned into the pDM327 vector, upstream from the bar-gus fusion gene for Biolistic™ transformation. Transformation of lupin embryos, bean callus tissue, maize embryos and Ornithogalum callus demonstrated promoter activity for all fragments. In silico analysis identified putative cis-acting elements in the 1.81 kb fragment that could be important in controlling gene expression. Fungal elicitor activated-, wound-inducible- and ethylene responsive elements were present in the 1.51 kb fragment. Myb elements and CAAT boxes that regulate responses to environmental factors and modulate promoter efficiency were identified in the 1.81 kb fragment. The 1.51 and 1.81 kb fragments were inserted upstream of the gus gene into the pBI121 vector for Agrobacterium tumefaciens transformation of tobacco. Quantitative GUS assays indicated that the promoter fragments are functional in planta and inducible by defense-related signals, wounding, as well as chemical elicitation. All important elements essential for Bion inducibility are present on the shorter (1.51 kb) promoter fragment, but both 5′ distal and proximal cis-elements are required for full functionality. The IF3 promoter is, thus, suitable for use in defense gene constructs prepared for the production of anthracnose resistant lupin.
KeywordsBiolistics Cis-elements Chitinase Lupin Pathogenesis-related Promoter Regulation
The financial assistance of the South African Agricultural Research Council (ARC) and the Protein Research Trust (PRT) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the authors and are not necessarily to be attributed to ARC or PRT. We thank L. Morey for assistance with the statistical analysis.
Compliance with ethical standards
Conflict of interest
The authors declare no competing interests.
- Adhikari, K. N., Thomas, G., Diepeveen, D., & Trethowan, R. (2013). Overcoming the barriers of combining early flowering and anthracnose resistance in white lupin (Lupinus albus L.) for the Northern agricultural region of Western Australia. Crop and Pasture Science, 64, 914–921.CrossRefGoogle Scholar
- Broglie, K. E., Gaynor, J. J., & Broglie, R. M. (1986). Ethylene-regulated gene expression: molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris. Proceedings of the National Academy of Sciences of the United States of America, 83, 6820–6824.CrossRefPubMedPubMedCentralGoogle Scholar
- Bustos, M. M., Guiltinan, M. J., Jordano, J. H., Begum, D., Kalkan, F. A., & Hall, T. C. (1989). Regulation of β-glucuronidase expression in transgenic tobacco plants by an A/T rich, cis-acting sequence found upstream of french bean β-phaseolin gene. The Plant Cell, 1, 839–853.PubMedPubMedCentralGoogle Scholar
- GenStat (2011) 64-bit Release 14.1 (PC/Windows 7) Copyright 2011, VSN International Ltd.Google Scholar
- Itzhaki, H., Maxson, J. M., & Woodson, W. R. (1994). N ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene. Proceedings of the National Academy of Sciences of the United States of America, 91, 8925–8929.CrossRefPubMedPubMedCentralGoogle Scholar
- Kessmann, H., Staub, T., Hofmann, C., Ahl Goy, P., Ward, E., Uknes, S., et al. (1993). Induced disease resistance by isonicotinic acid derivatives. Japan Journal of Pesticide Science, 10, 29–37.Google Scholar
- Koch, S. H., Ghebremariam, D. S., & Swart, W. J. (2002). Susceptibility of lupin cultivars to south African isolates of Colletotrichum gloeosporioides associated with lupin anthracnose. African Plant Protection, 8, 51–56.Google Scholar
- Legrand, M., Kauffmann, S., Geoffroy, P., & Fritig, B. (1987). Biological function of pathogenesis-related proteins: four tobacco pathogenesis-related proteins are chitinases. Proceedings of the National Academy of Sciences of the United States of America, 84, 6750–6754.CrossRefPubMedPubMedCentralGoogle Scholar
- Lescot, M., Dehais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., et al. (2002). PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for the in silico analysis of promoter sequences. Nucleic Acids Research, 30, 325–327.CrossRefPubMedPubMedCentralGoogle Scholar
- Neuhaus, J.-M. (1999). Plant chitinases (PR-3, PR-4, PR-8, PR-11). In S. K. Datta & S. Muthukrishnan (Eds.), Pathogenesis-related proteins in plants (pp. 77–105). Boca Raton, FL: CRC Press.Google Scholar
- Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning. A laboratory manual (2nd ed.). Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.Google Scholar