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


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.


Biolistics 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.

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)


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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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