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


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 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Al-Samarrai TH and Schmid J (2000) A simple method for extraction of fungal genomic DNA. Letters in Applied Microbiology 30: 53–56Google Scholar
  2. Al-Samarrai TH, Sullivan PA, Templeton MD and Farley PC (2002) Peptide inhibitors of appressorium development in Glomerella cingulata. FEMS Microbiology Letters 209: 203–207Google Scholar
  3. Apodaca G and McKerrow JH (1989) Purification and characterization of a 27,000-M r extracellular proteinase from Trichophyton rubrum. Infection and Immunity 57: 3072–3080Google Scholar
  4. Ball AM, Ashby AM, Daniels MJ, Ingram DS and Johnstone K (1991) Evidence for the requirement of extracellular proteinase in the pathogenic interaction of Pyrenopeziza brassicae with oilseed rape. Physiological and Molecular Plant Pathology 38: 147–161Google Scholar
  5. Bindschedler LV, Sanchez P, Dunn S, Mikan J, Thangavelu M, Clarkson JM and Cooper RM (2003) Deletion of the SNP1 trypsin protease from Stagonospora nodorum reveals another major protease expressed during infection. Fungal Genetics and Biology 38: 43–53Google Scholar
  6. Bowen JK, Templeton MD, Sharrock KR, Crowhurst RN and Rikkerink EHA (1995) Gene inactivation in the plant pathogen Glomerella cingulata: Three strategies for the disruption of the pectin lyase gene pnlA. Molecular and General Genetics 246: 196–205Google Scholar
  7. Carlile AJ, Bindschedler LV, Bailey AM, Bowyer P, Clarkson JM and Cooper RM (2000) Characterization of SNP1, a cell wall-degrading trypsin, produced during infection by Stagonospora nodorum. Molecular Plant-Microbe Interactions 13: 538–550Google Scholar
  8. Christeller JT, Farley PC, Ramsay RJ, Sullivan PA and Laing WA (1998) Purification, characterization and cloning of an aspartic proteinase inhibitor from squash phloem exudate. European Journal of Biochemistry 254: 160–167Google Scholar
  9. Church GM and Gilbert W (1984) Genomic sequencing. Proceedings of the National Academy of Science 81: 1991–1995Google Scholar
  10. Clark SJ, Templeton MD and Sullivan PA (1997) A secreted aspartic proteinase from Glomerella cingulata: Purification of the enzyme and molecular cloning of the cDNA. Microbiology 143: 1395–1403Google Scholar
  11. Cordero MJ, Raventos D and San Segundo B (1994) Expression of a maize proteinase inhibitor gene is induced in response to wounding and fungal infection: Systemic wound-response of a monocot gene. Plant Journal 6: 141–150Google Scholar
  12. Di Pietro A, Huertas-Gonzalez MD, Gutierrez-Corona JF, Martinez-Cadena G, Meglecz E and Roncero MIG (2001) Molecular characterization of a subtilase from the vascular wilt fungus Fusarium oxysporum. Molecular Plant-Microbe Interaction 14: 653–662Google Scholar
  13. DelMar EG, Largman C, Brodrick JW and Geokas MC (1979) A sensitive new substrate for chymotrypsin. Analytical Biochemistry 99: 316–320Google Scholar
  14. Froeliger EH and Carpenter BE (1996) NUT1, a major nitrogen regulatory gene in Magnaporthe grisea, is dispensable for pathogenicity. Molecular and General Genetics 251: 647–656Google Scholar
  15. Gorlach JM, van der Knaap E and Walton JD (1998) Cloning and targeted disruption of MLG1, gene encoding two of three extra-cellular mixed-linked glucanases of Cochliobolus carbonum. Applied and Environmental Microbiology 64: 385–391Google Scholar
  16. Iida H, Takeuchi M and Ichishima E (1988) Action of Aspergillus serine proteinase on fluorogenic and chromogenic substrates. Agricultural and Biological Chemistry 52: 1281–1282Google Scholar
  17. Irwin JAH and Cameron DF (1978) Two diseases of Stylosanthes spp. caused by Colletotrichum gloeosporioides in Australia and pathogen specialisation within one of the causal organisms. Australian Journal of Agricultural Research 29: 305–317Google Scholar
  18. Jara P, Gilbert S, Delmas P, Guillemot JC, Kaghad M, Ferrara P and Loison G (1996) Cloning and characterization of the eapB and eapC genes of Cryphonectria parasitica encoding two new acid proteinases, and disruption of eapC. Molecular and General Genetics 250: 97–105Google Scholar
  19. Joshi BN, Sainani MN, Bastawade JB, Gupta VS and Ranjekar PK (1998) Cysteine protease inhibitor from pearl millet: A new class of antifungal protein. Biochemical and Biophysical Research Communications 246: 382–387Google Scholar
  20. Kuć J and Williams EB (1962) Production of proteolytic enzymes by four pathogens of apple fruit. Phytopathology 52: 739Google Scholar
  21. Larcher G, Bouchara J-P, Annaix V, Symoens F, Chabasse D and Tronchin G (1992) Purification and characterization of a fibrinogenolytic serine proteinase from Aspergillus fumigatus culture filtrate. FEBS Letters 308: 65–69Google Scholar
  22. Larcher G, Cimon B, Symoens F, Tronchin G, Chabasse D and Bouchara JP (1996) A 33 kDa serine proteinase from Scedosporium apiospermum. Biochemical Journal 315: 119–126Google Scholar
  23. Lebrun-Garcia A, Bourque S, Binet MN, Ouaked F, Wendehenne D, Chiltz A, Schaffner A and Pugin A (1999) Involvement of plasma membrane proteins in plant defense responses. Analysis of the cryptogein signal transduction in tobacco. Biochimie 81: 663–668Google Scholar
  24. Mordue JEM (1971) Glomerella cingulata. CMI/AAB Descriptions of Pathogenic Fungi and Bacteria, No. 315Google Scholar
  25. Movahedi S and Heale JB (1990a) Purification and characterization of an aspartic proteinase secreted by Botrytis cinerea Pers ex. Pers in culture and in infected carrots. Physiological and Molecular Plant Pathology 36: 289–302Google Scholar
  26. Movahedi S and Heale JB (1990b) The roles of an aspartic proteinase and endo-pectin lyase enzymes in the primary stages of infection and pathogenesis of various host tissues by different isolates of Botrytis cinerea Pers ex. Pers. Physiological and Molecular Plant Pathology 36: 303–324Google Scholar
  27. Murphy JM and Walton JD (1996) Three extracellular proteases from Cochliobolus carbonum: Cloning and targeted disruption of ALP1. Molecular Plant-Microbe Interaction 9: 290–297Google Scholar
  28. Pekkarinen AI, Jones BL and Niku-Paavola ML (2002) Purification and properties of an alkaline proteinase of Fusarium culmorum. European Journal of Biochemistry 269: 798–807Google Scholar
  29. Pernas M, Sanchez-Monge R and Salcedo G (2000) Biotic and abiotic stress can induce cystatin expression in chestnut. FEBS Letters 467: 206–210Google Scholar
  30. Poussereau N, Creton S, Billon-Grand G, Rascle C and Fevre M (2001) Regulation of acp1, encoding a non-aspartyl acid protease expressed during pathogenesis of Sclerotinia sclerotiorum. Microbiology 147: 717–726Google Scholar
  31. Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH and van den Hondel CAMJJ (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56: 117–124Google Scholar
  32. Ramesh MV and Kolattukudy PE (1996) Disruption of the serine proteinase gene (sep) in Aspergillus flavus leads to a compensatory increase in the expression of a metalloproteinase gene (mep20). Journal of Bacteriology 178: 3899–3907Google Scholar
  33. Razanamparany V, Jara P, Legoux R, Delmas P, Msayeh F, Kaghad M and Loison G (1992) Cloning and mutation of the gene encoding endothiapepsin from Cryphonectria parasitica. Current Genetics 21: 455–461Google Scholar
  34. Redman RS and Rodriguez RJ (2002) Characterization and isolation of an extracellular serine protease from the tomato pathogen Colletotrichum coccodes, and its role in pathogenicity. Mycological Research 106: 1427–1434Google Scholar
  35. Redman RS, Ranson JC and Rodriguez RJ (1999) Conversion of the pathogenic fungus Colletotrichum magna to a nonpathogenic, endophytic mutalist by gene disruption. Molecular Plant-Microbe Interactions 12: 969–975Google Scholar
  36. Rikkerink EHA, Solon SL, Crowhurst RN and Templeton MD (1994) Integration of transformation vectors by homologous recombination in the plant pathogen Glomerella cingulata. Current Genetics 25: 202–208Google Scholar
  37. Roby D, Toppan A and Esquerre-Tugaye M-T (1987) Cell surfaces in plant micro-organism interactions. VIII. Increased proteinase inhibitor activity in melon plants in response to infection by Colletotrichum lagenarium or to treatment with an elicitor fraction from this fungus. Physiological and Molecular Plant Pathology 30: 453–460Google Scholar
  38. Rodriguez RJ (1993) Polyphosphate present in DNA preparations from filamentous fungal species of Colletotrichum inhibits restriction endonucleases and other enzymes. Analytical Biochemistry 209: 291–297Google Scholar
  39. Rogers LM, Kim YK, Guo W, Gonzalez-Candelas L, Li D and Kolattukudy PE (2000) Requirement for either a host-or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca. Proceedings of the National Academy of Science 97: 9813–9818Google Scholar
  40. Ryan CA (1990) Protease inhibitors in plants: Genes for improving defenses against insects and pathogens. Annual Review of Phytopathology 28: 425–429Google Scholar
  41. Sambrook J, Frisch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  42. Scott-Craig JS, Cheng YQ, Cervone F, de-Lorenzo G, Pitkin JW and Walton JD (1998) Targeted mutants of Cochliobolus carbonum lacking the two major extracellular polygalacturonases. Applied and Environmental Microbiology 64: 1497–1503Google Scholar
  43. Showalter AM (1993) Structure and function of plant cell wall proteins. The Plant Cell 5: 9–23Google Scholar
  44. Sreedhar L, Kobayashi DY, Bunting TE, Hillman BI and Belanger FC (1999) Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host. Gene 235: 121–129Google Scholar
  45. Templeton MD, Rikkerink EHA, Solon SL and Crowhurst RN (1992) Cloning and molecular characterisation of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata. Gene 122: 225–230Google Scholar
  46. van Kan JA, van't Klooster JW, Wagemakers CA, Dees van DC and der Vlugt-Bergmans CJ (1997) Cutinase A of Botrytis cinerea is expressed, but not essential, during penetration of gerbera and tomato. Molecular Plant-Microbe Interactions 10: 30–38Google Scholar
  47. Yakoby N, Beno-Moualem D, Keen NT, Dinoor A, Pines O and Prusky D (2001) Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction. Molecular Plant-Microbe Interactions 14: 988–995Google Scholar

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

Personalised recommendations