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

Wang X, Li Q, Niu X, Chen H, Xu L, Qi C - J. Exp. Bot. (2009)

Bottom Line: Two additional members of the IPG-1gene family were isolated by RT-PCR.Their sequence similarity with IPG-1 is as high as 95%.Possible roles of IPG-1 and its association with sspg1d in the defence signalling pathway were discussed.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China. xywang@njau.edu.cn

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.

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Localization of IPG-1-GFP fusion in onion epidermal cell. Plant cell wall and membrane were separated by treatment with 20% sugar.
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fig4: Localization of IPG-1-GFP fusion in onion epidermal cell. Plant cell wall and membrane were separated by treatment with 20% sugar.

Mentions: It is known that C2 domain proteins play a role in Ca2+-dependent spatio-temporal targeting in different regulatory signal transduction chains (Evans et al., 2004). Furthermore, it has been shown that the small rice C2 domain protein OsERG1 is translocated to the plasma membrane of plant cells in a Ca2+-dependent manner (Kim et al., 2003). To determine whether the newly identified C2 domain protein IPG-1 also exhibits calcium-dependent subcellular localization, the IPG-1–GFP construct driven by the CaMV 35S promoter was introduced into onion epidermal cells by bombardment for transient expression. As shown in Fig. 4, IPG-1 protein is mainly targeted to the plasma membrane and nucleus. After treatment with ionomycin, a calcium ionophore, for 5 h, the IPG-1–GFP signals was observed distributing throughout the cytosol (Fig. 5). It means that the Ca2+ ionophore treatment induced translocation of the green fluorescence signal emitted from the IPG-1-GFP from the plasma membrane and the nucleus to the cytosol.


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

Wang X, Li Q, Niu X, Chen H, Xu L, Qi C - J. Exp. Bot. (2009)

Localization of IPG-1-GFP fusion in onion epidermal cell. Plant cell wall and membrane were separated by treatment with 20% sugar.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2692008&req=5

fig4: Localization of IPG-1-GFP fusion in onion epidermal cell. Plant cell wall and membrane were separated by treatment with 20% sugar.
Mentions: It is known that C2 domain proteins play a role in Ca2+-dependent spatio-temporal targeting in different regulatory signal transduction chains (Evans et al., 2004). Furthermore, it has been shown that the small rice C2 domain protein OsERG1 is translocated to the plasma membrane of plant cells in a Ca2+-dependent manner (Kim et al., 2003). To determine whether the newly identified C2 domain protein IPG-1 also exhibits calcium-dependent subcellular localization, the IPG-1–GFP construct driven by the CaMV 35S promoter was introduced into onion epidermal cells by bombardment for transient expression. As shown in Fig. 4, IPG-1 protein is mainly targeted to the plasma membrane and nucleus. After treatment with ionomycin, a calcium ionophore, for 5 h, the IPG-1–GFP signals was observed distributing throughout the cytosol (Fig. 5). It means that the Ca2+ ionophore treatment induced translocation of the green fluorescence signal emitted from the IPG-1-GFP from the plasma membrane and the nucleus to the cytosol.

Bottom Line: Two additional members of the IPG-1gene family were isolated by RT-PCR.Their sequence similarity with IPG-1 is as high as 95%.Possible roles of IPG-1 and its association with sspg1d in the defence signalling pathway were discussed.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China. xywang@njau.edu.cn

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.

Show MeSH
Related in: MedlinePlus