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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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Expression of IPG-1 in different organs of canola analysed by real-time PCR with eukaryotic elongation factor 1-α (EF-1α) gene as internal control. (A) Without inoculation with Sclerotinia sclerotiorum. (B) Inoculation with Sclerotinia sclerotiorum.
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fig6: Expression of IPG-1 in different organs of canola analysed by real-time PCR with eukaryotic elongation factor 1-α (EF-1α) gene as internal control. (A) Without inoculation with Sclerotinia sclerotiorum. (B) Inoculation with Sclerotinia sclerotiorum.

Mentions: Semi-quantitative PCR revealed that IPG-1 was highly induced by Sclerotinia sclerotiorum in canola leaf and stem (Wang et al., 2008). Here, real-time PCR was used to analyse the expression of IPG-1 in leaf, stem, and flower organs of canola following inoculation with Sclerotinia sclerotiorum. As shown in Fig. 6, the expression of IPG-1 in flowers is about three times higher than that in leaves and stems before inoculating with Sclerotinia sclerotiorum, whereas the expression level of IPG-1 in leaves and stems was about 2–3 times higher than in flowers following inoculation with Sclerotinia sclerotiorum. The experiment again provided evidence that IPG-1 is significantly induced in leaves and stems by Sclerotinia sclerotiorum.


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)

Expression of IPG-1 in different organs of canola analysed by real-time PCR with eukaryotic elongation factor 1-α (EF-1α) gene as internal control. (A) Without inoculation with Sclerotinia sclerotiorum. (B) Inoculation with Sclerotinia sclerotiorum.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Expression of IPG-1 in different organs of canola analysed by real-time PCR with eukaryotic elongation factor 1-α (EF-1α) gene as internal control. (A) Without inoculation with Sclerotinia sclerotiorum. (B) Inoculation with Sclerotinia sclerotiorum.
Mentions: Semi-quantitative PCR revealed that IPG-1 was highly induced by Sclerotinia sclerotiorum in canola leaf and stem (Wang et al., 2008). Here, real-time PCR was used to analyse the expression of IPG-1 in leaf, stem, and flower organs of canola following inoculation with Sclerotinia sclerotiorum. As shown in Fig. 6, the expression of IPG-1 in flowers is about three times higher than that in leaves and stems before inoculating with Sclerotinia sclerotiorum, whereas the expression level of IPG-1 in leaves and stems was about 2–3 times higher than in flowers following inoculation with Sclerotinia sclerotiorum. The experiment again provided evidence that IPG-1 is significantly induced in leaves and stems by Sclerotinia sclerotiorum.

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