Characterization of a canola C2 domain gene that interacts with PG, an effector of the necrotrophic fungus Sclerotinia sclerotiorum

Sspg1d, one of endopolygalacturonases, is an important fungal effector secreted by the necrotrophic fungus Sclerotinia sclerotiorum during early infection. Using sspg1d as bait, a small C2 domain protein (designated as IPG-1) was identified by yeast two-hybrid screening of a canola cDNA library. Deletion analysis confirmed that the C-terminus of IPG-1 is responsible for its interaction with sspg1d in the yeast two-hybrid assay. The sspg1d/IPG-1 interaction was further confirmed in plant cells by a biomolecular fluorescence complementation (BiFC) assay. A transient expression assay showed that the IPG-1-GFP fusion protein was targeted to the plasma membrane and nucleus in onion epidermal cells. Following treatment with a Ca2+ ionophore, it was distributed throughout the cytosol. Real-time PCR assay demonstrated that IPG-1 was highly induced by Sclerotinia sclerotiorum in canola leaves and stems. Southern blot analysis indicated the presence of about five homologues of IPG-1 in the canola genome. Two additional members of the IPG-1gene family were isolated by RT-PCR. Their sequence similarity with IPG-1 is as high as 95%. However, they did not interact with sspg1d in the yeast two-hybrid assay. Possible roles of IPG-1 and its association with sspg1d in the defence signalling pathway were discussed.