BIOCHEMICAL ANALYSIS OF THE INTERACTION BETWEEN VENTURIA INAEQUALIS, THE APPLE SCAB PATHOGEN, AND ITS HOST MALUS SP.
SJ COOPER and AM ASHBY
Department of Plant Sciences, University of Cambridge, Downing Site, Cambridge CB2 3EA, UK
Background and objectives
Symptoms such as green islands and leaf curl are thought to be caused by plant growth regulators. Cytokinins have been shown to be produced by fungi, although there is no direct molecular evidence to support a role for these in pathogenesis by fungi. We wish to establish if fungally derived cytokinins and fungal beta-glucosidases are capable of releasing cytokinins from their stored forms in early pathogenesis. By comparing results for Venturia inaequalis with those for other plant pathogens we are looking to investigate whether there is any correlation between production of cytokinins and cytokinin-releasing beta-glucosidases and the mode of nutrition displayed by the fungus in planta.
Materials and methods
Cytokinin production was assayed by HPLC-ELISA. Methanolic extracts of fungi grown on rich and minimal media were run on a 15-36% methanol gradient through a C18 column and fractions collected. Fractions were subjected to competitive ELISA  to measure the levels of cytokinins produced. Cytokinin O-linked and N-linked glucosides were used as substrates to assess cytokinin glucoside-cleaving ability of crude protein extracts from a range of plant pathogens grown under differing conditions. HPLC and TLC were used to analyse the extracts 1,5 glucono-d-lactone was used as a competitive inhibitor to confirm beta-glucosidase activity.
Results and conclusions
Levels of cytokinins from mycelium of Venturia inaequalis were significantly higher for samples from the minimal (Murashige and Skoog, glucose and vitamins) media (zeatin riboside equivalent 264.2 pmol/g fw) than for samples from the rich (malt extract and salts) media (zeatin riboside equivalent 43.7 pmol/g fw). beta-Glucosidases capable of releasing cytokinins from their O-linked, but not N-linked glucosides were found in crude protein extracts for V. inaequalis grown on both rich and minimal media. Values for zeatin-o-glucoside-cleaving activity were compared with those for a range of other plant pathogenic fungi with differing modes of nutrition in planta. This is the first report of cytokinin-glucoside-cleaving beta-glucosidase activity in a filamentous fungus, and possible roles for this fungal derived enzyme in plant nutrition will be presented.
1. Huntley RP, 1995. PhD thesis, University of Cambridge, UK.