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Title: Cytokinin and cytokinin-glucoside cleaving beta-glucosidase production by Venturia inequalis
Author: Cooper, S. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1999
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To localise cytokinins in V. inaequalis infected apple tissue immuno-fluorescence microscopy was employed and demonstrated that cytokinins localised around the V. inaequalis hyphae. PCR and Southern analysis were used in an attempt to identify the gene(s) involved in cytokinin production by V. inaequalis. Cytokinin production was also confirmed for the light leaf spot pathogen of brassicas. Pyrenopeziza brassicae, using HPLC-ELISA. Another plant pathogen, Leptoxphaeria maculans, appeared not to be producing cytokinins under the conditions tested. Cytokinin-O-glucoside cleaving β-glucosidase activity that released free base zeatin from zeatin-O-glucoside was identified for the first time in plant pathogenic fungi. In V. inaequalis cytokinin-O-glucoside cleaving β-glucosidase activity was found to significantly increase, relative to total β-glucosidase activity was found to significantly increase, relative to total β-glucosidase activity, when the fungus was grown under growth limiting instead of nutrient rich-conditions in vitro. A protocol was developed for the purification of the enzyme that produced this activity and it was purified to apparent homogeneity. Attempts were made to gain a handle on the gene(s) involved in producing this activity using heterologous Southern hybridisation to known cytokinin glucoside cleaving β-glucosidases from plants. In addition cytokinin receptors were sought from apple in order to provide a means to assess fungal cytokinin production in planta. Genes might be involved in the biotrophic phase of V. inaequalis infection were sought by constructing a genomic library of V. inaequalis. The library was differentially screened with 1st strand cDNAs derived from V. inaequalis grown under growth limiting and nutrient rich conditions to identify putative fungal genes expressed in planta. The findings of this study were related to the modes of nutrition of the plant pathogens studied. It emerged that, in general, cytokinin production and cytokinin-O-glucoside cleaving β-glucosidase activity were a feature of biotrophic and hemibiotrophic fungi but not necrotrophic fungi.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available