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European Journal of Plant Pathology

, Volume 110, Issue 3, pp 265–274 | Cite as

Analysis of a Secreted Aspartic Peptidase Disruption Mutant of Glomerella cingulata

  • Kim M. Plummer
  • Sarah J. Clark
  • Lana M. Ellis
  • Ashwini Loganathan
  • Taha H. Al-Samarrai
  • Erik H.A. Rikkerink
  • Patrick A. Sullivan
  • Matthew D. Templeton
  • Peter C. Farley
Article

Abstract

Peptidases have been implicated in the pathogenicity of fungi that cause disease in plants. Expression of the secreted aspartic peptidase gene (gcsap), of a Glomerella cingulata isolate pathogenic on apples, is induced during appressorium formation. To determine whether the secreted aspartic peptidase (GcSAP) is essential to pathogenicity, gcsap was disrupted using a vector containing a 637 bp fragment of genomic DNA that encodes the sequence spanning the two active site aspartic acid (Asp) residues. To ensure that the truncated gcsap gene products could not have residual peptidase activity the codons for the active site residues Asp112 and Asp297 were both mutated to histidine residues. Both PCR and Southern analysis confirmed disruption of gcsap. Neither gcsap mRNA nor GcSAP activity was detected in the disruption mutant. Pathogenicity tests on fruit from three apple cultivars showed that GcSAP was not required for pathogenicity. The disruption mutant grew on medium containing protein as the sole source of nitrogen because G. cingulata secretes a previously undetected peptidase(s). A serine peptidase that had a pH optimum between pH 7.0 and 8.0 and a Km of 0.25 mM for the synthetic substrate succinyl-Ala–Ala–Pro–Phe-p-nitroanilide was identified.

aspartic peptidase gene disruption Glomerella cingulata Malus domestica serine peptidase phytopathogen 

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Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Kim M. Plummer
    • 1
  • Sarah J. Clark
    • 2
  • Lana M. Ellis
    • 2
  • Ashwini Loganathan
    • 3
  • Taha H. Al-Samarrai
    • 3
  • Erik H.A. Rikkerink
    • 4
  • Patrick A. Sullivan
    • 3
  • Matthew D. Templeton
    • 4
  • Peter C. Farley
  1. 1.School of Biological SciencesThe University of AucklandAucklandNew Zealand
  2. 2.Biochemistry DepartmentThe University of OtagoDunedinNew Zealand
  3. 3.Institute of Molecular Biosciences, PN462Massey UniversityPalmerston NorthNew Zealand
  4. 4.Plant Health and Development UnitHorticulture and Food Research Institute of New ZealandAucklandNew Zealand

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