The extracellular protease (PEP) of <I>Pyrenopeziza brassicae</I>, an economically important pathogen of oilseed rape and other brassicas, may be a significant factor in the development of the light leaf spot disease. The aim of this study was to further our understanding of PEP and the role of this enzyme in the disease process. Results of purification of PEP, monitored with a new microtite plate assay based on chromogenic protease substrates, are presented. The partial N-terminal sequence of the mature enzyme was reconfirmed and shown to share high levels of homology with fungal extracellular serine proteases of the subtilisin (S8) family. Internal amino acid sequence was also obtained from purified PEP. PEP was characterised by its inhibitor mixed trypsin/chymotrypsin specificity. PEP has a K<I><SUB>m</SUB></I> for a trypsin substrate that is the same as that for trypsin. In order to isolate the cDNA clone of the structural gene of PEP, a skimmed milk-induced cDNA library was screened with two separate probes. The first was a reverse transcription-polymerase chain reaction (RT-PCR) product amplified from total mycelial RNA with a primer designed from the N-terminal amino acid sequence of PEP and a poly-T primer. The second was a PCR product amplified from genomic DNA by primers designed from the PEP internal amino acid sequence. Although several cDNA clones were isolated by these approaches, none were identified as corresponding to PEP. Finally, antigens were identified in the epidermal cell walls of a light leaf spot susceptible cultivar of oilseed rape which may represent potential glycoprotein targets for PEP activity <I>in planta</I>. The antigens were recognised by antibodies that recognise the sugar moiety of turnip antigens, one of which is postulated to have a role in the progress of black rot disease caused by <I>Xanthomonas campestris </I>pv. <I>campestris</I> due to its susceptibility to degradation by an extracellular protease produced by that pathogen <I>in planta</I> (Davies 1996).
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:604806 |
| Date | January 1997 |
| Creators | Hunter, A. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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