title: Characterization of a canola C2 domain gene that interacts with PG, an effector of the necrotrophic fungus Sclerotinia sclerotiorum.
abstract: 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 Ca(2+) 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.
authors.affiliation: College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China. xywang@njau.edu.cn
authors.forename: Xinyu
authors.initials: X
authors.lastname: Wang
authors.forename: Qian
authors.initials: Q
authors.lastname: Li
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Niu
authors.forename: Haiyan
authors.initials: H
authors.lastname: Chen
authors.forename: Langlai
authors.initials: L
authors.lastname: Xu
authors.forename: Cunkou
authors.initials: C
authors.lastname: Qi
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000235:genetics / Q000378:metabolism*; D029688:Brassica napus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011096:Polygalacturonase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques
chemical_list: D005656:Fungal Proteins; D010940:Plant Proteins; D011096:Polygalacturonase