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 suspected to play an important role in plant defense. Several phytopathogenic fungi are able to detoxify these compounds by deglycosylation, and this ability could be a factor of general importance for the successful colonization of saponin-containing plants. Botrytis cinerea, causing 'grey mould' of many (especially greenhouse) crops and vegetables, among them several plants containing saponins, has been reported to deglycosylate and detoxify alpha-tomatine. Here we show that it can also degrade avenacin, avenacosides and digitonin, and that it possesses more than one saponin-specific enzyme. Using the tomatinase gene (tom1) from Septoria lycopersici as probe, we have cloned and characterized a gene (sap1) coding for a 83 kDa polypeptide which shows significant homology to tom1 and to the avenacinase gene of Gaeumannomyces graminis. Disruption of sap1 leads to loss of the ability to deglycosylate avenacin, whereas sap1 deletion mutants still can detoxify tomatine, digitonin and avenacosides, i.e. sap1 encodes an 'avenacinase'. The putative 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 against avenacosides. Since a tomatinase-deficient field isolate of B. cinerea, M3, also lacks the ability to deglycosylate digitonin, but can degrade avenacin and avenacosides, these data confirm the existence of at least three distinct activities of saponin-specific glycosidases in B. cinerea: a xylosidase (alpha-tomatinase, digitoninase), and two different glucosidases (avenacinase and avenacosidase).