Endopolygalacturonases from Botrytis cinerea: biochemical properties and interaction with inhibiting proteins

The phytopathogen Botrytis cinerea harbours at least six endopolygalacturonase-encoding (Bcpg) genes of which Bcpg1 and Bcpg2 are required for full virulence. The endopolygalacturonase (BcPG) enzymes degrade pectin, enabling the fungus to breach the plant cell wall. We expressed five BcPG isozymes in Pichia pastoris, purified them and studied biochemical properties, such as pH optimum, mode of action, and substrate specificity in detail. BcPG3 shows a rather unusual, broad pH optimum and is the only isozyme fully active at pH 3.5. Polygalacturonic acid is a poor substrate for BcPG1 and BcPG4 as compared to BcPG2, BcPG3, and BcPG6. In contrast to BcPG1, 2, and 4, BcPG3 and BcPG6 show extreme processive behaviour on oligogalacturonides longer than four GalpA residues. Only BcPG3 and BcPG6 are able to hydrolyse GalpA dimers. Since PG activity is important for fungal virulence, the BcPGs are interesting targets for disease control. Therefore, a range of plant extracts was screened to identify potent polygalacturonase-inhibiting proteins (PGIPs). PGIPs from different plant sources have been purified and their interaction with the BcPG isozymes have been investigated using different techniques. Results on the mode of inhibition and binding will be presented.