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European Journal of Plant Pathology

, Volume 146, Issue 4, pp 923–936 | Cite as

Functional characterization of a defense-related class-III chitinase promoter from Lupinus albus, active in legume and monocot tissues

  • Dean Oelofse
  • Inge Gazendam
  • Adri Veale
  • Arnaud Djami-Tchatchou
  • Dave Berger
  • Ian DuberyEmail author
Article

Abstract

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.

Keywords

Biolistics Cis-elements Chitinase Lupin Pathogenesis-related Promoter Regulation 

Notes

Acknowledgments

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.

Supplementary material

10658_2016_970_MOESM1_ESM.pdf (308 kb)
Figure S1 (PDF 308 kb)
10658_2016_970_MOESM2_ESM.docx (26 kb)
Figure S2 A graphic representation of the 3′ section of the 4.2 kb class-III chitinase (IF3) promoter-containing fragment isolated from Lupinus albus. Following promoter prediction analysis, the 2.18 kb fragment obtained from the pGEM:2.2Promoter was cut with restriction enzymes to yield 1.81 kb and 1.51 kb fragments (each containing three predicted core promoter elements: P1, P2 and P3), and a 1.13 kb fragment (containing 2 predicted core promoter sequences: P1 and P2). The score values of P1, P2 and P3 were 0.88, 0.81 and 0.81 respectively. (DOCX 25 kb)
10658_2016_970_MOESM3_ESM.docx (62 kb)
Figure S3 Restriction enzyme digests of the pGEM:2.2Promoter for the creation of constructs corresponding to 1.81, 1.51 and 1.13 kb class-III chitinase (IF3) promoter-containing fragments, respectively. Lane 1: Molecular Weight Marker III (Roche); Lane 2: uncut pGEM:2.2Promoter clone; Lane 3: NcoI/BamHI digest; Lane 4: NsiI/BamHI digest; Lane 5: BglII/BamHI digest. The arrows indicate the expected 1.81, 1.51 and 1.13 kb IF3 promoter-containing fragments. (DOCX 62 kb)
10658_2016_970_MOESM4_ESM.pdf (76 kb)
Figure S4 (PDF 76 kb)

References

  1. Adhikari, K. N., Buirchell, B. J., Thomas, G. J., Sweetingha, M. W., & Yang, H. (2009). Identification of anthracnose resistance in Lupinus albus L. and its transfer from landraces to modern cultivars. Crop and Pasture Science, 60, 472–479.CrossRefGoogle Scholar
  2. 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
  3. Azhakanandam, K., Silverstone, A., Daniell, H., & Davey, M. R. (Eds.) (2015). Recent advancements in gene expression and enabling technologies in crop plants (pp. 1–422). New York: Springer Verlag publishers.CrossRefGoogle Scholar
  4. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.CrossRefPubMedGoogle Scholar
  5. Broekaert, W. F., Van Parijs, J., Allen, A. K., & Peumans, W. J. (1988). Comparison of some molecular, enzymatic and antifungal properties of chitinases from thorn-apple, tobacco and wheat. Physiological and Molecular Plant Pathology, 33, 319–331.CrossRefGoogle Scholar
  6. 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
  7. Broglie, K., Chet, I., Holliday, M., Cressman, R., Biddle, P., Knowlton, S., et al. (1991). Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science, 254, 1194–1197.CrossRefPubMedGoogle Scholar
  8. 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
  9. Chang, W. C., Lee, T. Y., Huang, H. D., Huang, H. Y., & Pan, R. L. (2008). PlantPAN: plant promoter analysis navigator, for identifying combinatorial cis-regulatory elements with distance constraint in plant gene groups. BMC Genomics, 9, 561.CrossRefPubMedPubMedCentralGoogle Scholar
  10. Collinge, D. B., Kragh, K. M., Mikkelsen, I. D., Nieler, K. K., Rasmussen, U., & Vad, K. (1993). Plant chitinases. Plant Journal, 3, 31–40.CrossRefPubMedGoogle Scholar
  11. Cornejo, M. J., Luth, D., Blankenship, K. M., Anderson, O. D., & Blechl, A. E. (1993). Activity of a maize ubiquitin promoter in transgenic rice. Plant Molecular Biology, 23, 567–581.CrossRefPubMedGoogle Scholar
  12. De Villiers, S. M., Kamo, K., Thomson, J. A., Bornman, C. H., & Berger, D. K. (2001). Biolistic transformation of chincherinchee (Ornithogalum) and regeneration of transgenic plants. Physiologia Plantarum, 109, 450–455.CrossRefGoogle Scholar
  13. Ernst, D., Schraudner, M., Langebartels, C., & Sandermann, H. (1992). Ozone induced changes in mRNA levels of β-1,3-glucanase, chitinase and pathogenesis-related protein 1b in tobacco plants. Plant Molecular Biology, 20, 673–682.CrossRefPubMedGoogle Scholar
  14. Friedrich, L., Lawton, K., Reuss, W., Masner, P., Specker, N., Gut Rella, M., et al. (1996). A benzothiadiazole induces systemic acquired resistance in tobacco. Plant Journal, 10, 61–70.CrossRefGoogle Scholar
  15. GenStat (2011) 64-bit Release 14.1 (PC/Windows 7) Copyright 2011, VSN International Ltd.Google Scholar
  16. Graham, L. S., & Sticklen, M. B. (1994). Plant chitinases. Canadian Journal of Botany, 72, 1057–1083.CrossRefGoogle Scholar
  17. Grover, A. (2012). Plant chitinases: genetic diversity and physiological roles. Critical Reviews in Plant Sciences, 31, 57–73.CrossRefGoogle Scholar
  18. Gurr, S. J., & Rushton, P. J. (2005). Engineering plants with increased disease resistance: how are we going to express it? Trends in Biotechnology, 23, 283–290.CrossRefPubMedGoogle Scholar
  19. Henrissat, B., & Bairoch, A. (1993). New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal, 293, 781–788.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Higo, K., Ugawa, Y., Iwamoto, M., & Korenaga, T. (1999). Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Research, 27, 297–300.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 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
  22. Jach, G., Gornhardt, B., Mundy, J., Logemann, J., Pinsdorf, E., Leah, R., et al. (1995). Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant Journal, 8, 97–109.CrossRefPubMedGoogle Scholar
  23. Jefferson, R. A., Kavanagh, T. A., & Bevan, M. W. (1987). GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal, 6, 3901–3907.PubMedPubMedCentralGoogle Scholar
  24. Jin, H., & Martin, C. (2000). Multifunctionality and diversity within the plant MYB-gene family. Nucleic Acids Research, 28, 2004–2011.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kamo, K., McElroy, D., & Chamberlain, D. (2000). Transforming embryonic cell lines of Gladiolus with either a bar-uidA fusion gene or cobombardment. In Vitro Cellular and Developmental Plant Biology, 36, 182–187.CrossRefGoogle Scholar
  26. Kaothien, P., Shimokawatoko, Y., Kawaoka, A., Yoshida, K., & Shinmyo, A. (2000). A cis-element containing PAL-box functions in the expression of the wound-inducible peroxidase gene of horseradish. Pant Cell Reports, 19, 558–562.CrossRefGoogle Scholar
  27. 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
  28. 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
  29. Lawton, K. A., Beck, J., Potter, S., Ward, E., & Ryals, J. (1994). Regulation of cucumber class III chitinase gene expression. Molecular Plant-Microbe Interactions, 7, 335–341.CrossRefGoogle Scholar
  30. 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
  31. 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
  32. Lois, R., Dietrich, A., Hahlbrock, K., & Schulz, W. (1989). A phenylalanine ammonia-lyase gene from parsley: structure, regulation, and identification of elicitor and light responsive cis-acting elements. EMBO Journal, 8, 1641–1648.PubMedPubMedCentralGoogle Scholar
  33. Lotter, H. C., & Berger, D. K. (2005). Anthracnose of lupins in South Africa in caused by Colletotrichum lupini var. setosum. Australasian Plant Pathology, 34, 385–392.CrossRefGoogle Scholar
  34. Margis-Pinheiro, M., Martin, C., Didierjean, L., & Burkard, G. (1993). Differential expression of bean chitinase genes by virus infection, chemical treatment and UV irradiation. Plant Molecular Biology, 22, 659–668.CrossRefPubMedGoogle Scholar
  35. Matton, D. P., Prescott, G., Bertrand, C., Camirand, A., & Brisson, N. (1993). Identification of cis-acting elements involved in the regulation of the pathogenesis-related gene STH-2 in potato. Plant Molecular Biology, 22, 279–291.CrossRefPubMedGoogle Scholar
  36. Mauch, F., Mauch-Mani, B., & Boller, T. (1988). Antifungal hydrolases in pea tissue. II. Inhibition of funga1 growth by combinations of chitinase and P-1,3-glucanase. Plant Physiology, 88, 936–942.CrossRefPubMedPubMedCentralGoogle Scholar
  37. Memelink, J., Linthorst, H. J. M., Schilperpoort, R. A., & Hoge, J. H. C. (1990). Tobacco genes encoding acidic and basic isoforms of pathogenesis-related proteins display different expression patterns. Plant Molecular Biology, 14, 119–126.CrossRefPubMedGoogle Scholar
  38. Murray, M. G., & Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8, 4321–4325.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 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
  40. New, S.-A., Piater, L. A., & Dubery, I. A. (2015). In silico characterization and expression analysis of selected Arabidopsis receptor-like kinase genes responsive to different MAMP inducers. Biologia Plantarum, 59, 18–28.CrossRefGoogle Scholar
  41. Nirenberg, H. I., Feiler, U., & Hagedorn, G. (2002). Description of Colletotrichum lupini comb. nov. in modern terms. Mycologia, 94, 307–320.CrossRefPubMedGoogle Scholar
  42. Odell, J. T., Nagy, F., & Chua, N.-H. (1985). Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature, 313, 810–812.CrossRefPubMedGoogle Scholar
  43. Ohl, S., Hedrick, S. A., Chory, J., & Lamb, C. (1990). Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis. The Plant Cell, 2, 837–848.CrossRefPubMedPubMedCentralGoogle Scholar
  44. Pastuglia, M., Roby, D., Dumas, C., & Cock, J. M. (1997). Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase gene in Brassica oleracea. The Plant Cell, 9, 49–60.CrossRefPubMedPubMedCentralGoogle Scholar
  45. Porto, M. S., Pinheiro, M. P. N., Batista, V. G. L., dos Santos, R. C., Filho, P. A. M., & de Lima, L. M. (2014). Plant promoters: an approach of structure and function. Molecular Biotechnology, 56, 38–49.CrossRefPubMedGoogle Scholar
  46. Regalado, A. P., Pinheiro, C., Vidal, S., Chaves, I., Ricardo, C. P. P., & Rodrigues-Pousada, C. (2000). The Lupinus albus class-III chitinase gene, IF3, is constitutively expressed in vegetative organs and developing seeds. Planta, 210, 543–550.CrossRefPubMedGoogle Scholar
  47. Roby, D., Broglie, K., Cressman, R., Biddle, P., Chet, I., & Broglie, R. (1990). Activation of a bean chitinase promoter in transgenic tobacco plants by phytopathogenic fungi. Plant Cell, 2, 999–1008.CrossRefPubMedPubMedCentralGoogle Scholar
  48. Rushton, P. J., & Somssich, I. E. (1998). Transcriptional control of plant genes responsive to pathogens. Current Opinion in Plant Biology, 1, 311–315.CrossRefPubMedGoogle Scholar
  49. Rushton, P. J., Torres, J. T., Parniske, M., Wernert, P., Hahlbrock, K., & Somssich, I. E. (1996). Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO Journal, 15, 5690–5700.PubMedPubMedCentralGoogle Scholar
  50. Samac, D. A., & Shah, D. M. (1991). Developmental and pathogen-induced activation of the Arabidopsis acidic chitinase promoter. The Plant Cell, 3, 1063–1072.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 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
  52. Siebertz, B., Logemann, J., Willmitzer, L., & Schell, J. (1989). Cis-analysis of the wound-inducible promoter wun1 in transgenic tobacco plants and histochemical localization of its expression. The Plant Cell, 1, 961–968.CrossRefPubMedPubMedCentralGoogle Scholar
  53. Stanford, A. C., Bevan, M. H., & Northcote, D. H. (1989). Differential expression within a family of novel wound-induced genes in potato. Molecular and General Genetics, 215, 200–208.CrossRefPubMedGoogle Scholar
  54. Sugimoto, K., Takeda, S., & Hirochika, H. (2000). MYB-related transcription factor NtMYB2 induced by wounding and elicitors is a regulator of the tobacco retrotransposon Tto1 and defense-related genes. The Plant Cell, 12, 2511–2528.CrossRefPubMedPubMedCentralGoogle Scholar
  55. Thomasset, B., Menard, M., Boetti, H., Denmat, L. A., Inze, D., & Thomas, D. (1996). β-Glucuronidase activity in transgenic and non-transgenic tobacco cells: specific elimination of plant inhibitors and minimization of endogenous GUS background. Plant Science, 113, 209–219.CrossRefGoogle Scholar
  56. Verberg, J. G., & Huynh, Q. K. (1991). Purification and characterization of an antifungal chitinase from Arabidopsis thaliana. Plant Physiology, 95, 450–455.CrossRefGoogle Scholar
  57. Wang, Y.-C., Klein, T. M., Fromm, M., Cao, J., Sanford, J. C., & Wu, R. (1988). Transient expression of foreign genes in rice, wheat and soybean cells following particle bombardment. Plant Molecular Biology, 11, 433–439.CrossRefPubMedGoogle Scholar
  58. Ward, E. R., Uknes, S. J., Williams, S. C., Dincher, S. S., Wiederhold, D. L., Alexander, D. C., et al. (1991). Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell, 3, 1085–1094.CrossRefPubMedPubMedCentralGoogle Scholar
  59. Yeboah, N. A., Arahira, M., Nong, V. H., Zhang, D., Kadokura, K., Watanabe, A., et al. (1998). A class III acidic endochitinase is specifically expressed in the developing seeds of soybean (Glycine max L. Merr.). Plant Molecular Biology, 36, 407–415.CrossRefPubMedGoogle Scholar
  60. Zheng, Z., Kawagoe, Y., Xiao, S., Li, Z., Okita, T., Hau, T. L., et al. (1993). 5′ distal and proximal cis-acting regulator elements are required for developmental control of a rice seed storage protein glutelin gene. Plant Journal, 4, 357–366.CrossRefPubMedGoogle Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2016

Authors and Affiliations

  • Dean Oelofse
    • 1
    • 2
  • Inge Gazendam
    • 1
  • Adri Veale
    • 1
  • Arnaud Djami-Tchatchou
    • 2
  • Dave Berger
    • 3
  • Ian Dubery
    • 2
    Email author
  1. 1.Agricultural Research Council – Vegetable and Ornamental Plants (ARC-VOP)PretoriaSouth Africa
  2. 2.Department of BiochemistryUniversity of JohannesburgJohannesburgSouth Africa
  3. 3.Department of Plant Science, Forestry and Agricultural Biotechnology Institute (FABI)University of PretoriaPretoriaSouth Africa

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