Evidence for three different specific saponin-detoxifying activities in Botrytis cinerea and cloning and functional analysis of a gene coding for a putative avenacinase

Saponins are typical phytoanticipins, i.e. preformed fungitoxic compounds s uspected to play an important role in plant defense. Several phytopathogeni c fungi are able to detoxify these compounds by deglycosylation, and this a bility could be a factor of general importance for the successful colonizat ion of saponin-containing plants. Botrytis cinerea, causing 'grey mould' of many (especially greenhouse) crops and vegetables, among them several plan ts containing saponins, has been reported to deglycosylate and detoxify alp ha-tomatine. Here we show that it can also degrade avenacin, avenacosides a nd digitonin, and that it possesses more than one saponin-specific enzyme. Using the tomatinase gene (tom1) from Septoria lycopersici as probe, we hav e cloned and characterized a gene (sap1) coding for a 83 kDa polypeptide wh ich shows significant homology to tom1 and to the avenacinase gene of Gaeum annomyces graminis. Disruption of sap1 leads to loss of the ability to degl ycosylate avenacin, whereas sap1 deletion mutants still can detoxify tomati ne, digitonin and avenacosides, i.e. sap1 encodes an 'avenacinase'. The put ative product of this gene has been purified and characterized: it has a MW of 90,000, an isoelectric point (IP) of 5.2, and it has no activity agains t avenacosides. Since a tomatinase-deficient field isolate of B. cinerea, M 3, also lacks the ability to deglycosylate digitonin, but can degrade avena cin and avenacosides, these data confirm the existence of at least three di stinct activities of saponin-specific glycosidases in B. cinerea: a xylosid ase (alpha-tomatinase, digitoninase), and two different glucosidases (avena cinase and avenacosidase).