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Analysis of a Secreted Aspartic Peptidase Disruption Mutant of Glomerella cingulata

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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 K m of 0.25 mM for the synthetic substrate succinyl-Ala–Ala–Pro–Phe-p-nitroanilide was identified.

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Authors and Affiliations

  1. School of Biological Sciences, The University of Auckland, Auckland, New Zealand

    Kim M. Plummer

  2. Biochemistry Department, The University of Otago, Dunedin, New Zealand

    Sarah J. Clark & Lana M. Ellis

  3. Institute of Molecular Biosciences, PN462, Massey University, Private Bag 11222, Palmerston North, New Zealand

    Ashwini Loganathan, Taha H. Al-Samarrai & Patrick A. Sullivan

  4. Plant Health and Development Unit, Horticulture and Food Research Institute of New Zealand, Auckland, New Zealand

    Erik H.A. Rikkerink & Matthew D. Templeton

Authors
  1. Kim M. Plummer
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  2. Sarah J. Clark
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  3. Lana M. Ellis
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  4. Ashwini Loganathan
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Plummer, K.M., Clark, S.J., Ellis, L.M. et al. Analysis of a Secreted Aspartic Peptidase Disruption Mutant of Glomerella cingulata . European Journal of Plant Pathology 110, 265–274 (2004). https://doi.org/10.1023/B:EJPP.0000019796.78598.8c

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  • DOI: https://doi.org/10.1023/B:EJPP.0000019796.78598.8c

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