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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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Bimolecular fluorescence complementation assays in onion epidermal cells. The reconstitute YFP signals show that IPG-1 and sspg1d can associate in plant cells.
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fig3: Bimolecular fluorescence complementation assays in onion epidermal cells. The reconstitute YFP signals show that IPG-1 and sspg1d can associate in plant cells.

Mentions: As shown in Fig. 3, YFP fluorescence could be detected in onion epidermal cells co-transformed with YFPN–sspg1d and YFPC–IPG-1. No YFP fluorescence was detected in the negative controls (i.e. transformed with YFPC–IPG-1/YFPN, YFPN–IPG-1/YFPC) (data not shown). These results confirm that sspg1d interacts with IPG-1 in living plant cells.


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)

Bimolecular fluorescence complementation assays in onion epidermal cells. The reconstitute YFP signals show that IPG-1 and sspg1d can associate in plant cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Bimolecular fluorescence complementation assays in onion epidermal cells. The reconstitute YFP signals show that IPG-1 and sspg1d can associate in plant cells.
Mentions: As shown in Fig. 3, YFP fluorescence could be detected in onion epidermal cells co-transformed with YFPN–sspg1d and YFPC–IPG-1. No YFP fluorescence was detected in the negative controls (i.e. transformed with YFPC–IPG-1/YFPN, YFPN–IPG-1/YFPC) (data not shown). These results confirm that sspg1d interacts with IPG-1 in living plant cells.

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