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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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Amino acid sequence alignment of IPG-1 with its homologues BnC2d1 and BnC2d2. (This figure is available in colour at JXB online.)
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fig8: Amino acid sequence alignment of IPG-1 with its homologues BnC2d1 and BnC2d2. (This figure is available in colour at JXB online.)

Mentions: The copy numbers of IPG-1 in the canola genome were analysed by Southern blot of canola genomic DNA probed with IPG-1 cDNA (Fig. 7). The results showed that IPG-1 has about 2–5 copies in the canola genome, which means that the canola genome may have a C2 domain gene family. To test whether other members in the gene family interact with sspg1d, an additional two genes in the gene family were isolated by RT-PCR and designated as BnC2d1 and BnC2d2. BLAST analysis indicated that the two genes also contain the C2 domain. Sequence comparison of the two genes with IPG-1 revealed that their identity is as high as 95% in amino acids (Fig. 8). The coding sequences of BnC2d1 and BnC2d2 were individually fused into the GAL4 activating domain of the pGADT7 vector. Yeast mating was used to test the interaction between sspg1d and the two genes. To our surprise, the two genes did not interact with sspg1d, although the experiment was replicated several times. This means that the two IPG-1 homologues may have evolved functions different from IPG-1. This may explain why only one gene that interacts with sspg1d in the canola cDNA library was isolated by yeast two-hybrid screening.


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)

Amino acid sequence alignment of IPG-1 with its homologues BnC2d1 and BnC2d2. (This figure is available in colour at JXB online.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: Amino acid sequence alignment of IPG-1 with its homologues BnC2d1 and BnC2d2. (This figure is available in colour at JXB online.)
Mentions: The copy numbers of IPG-1 in the canola genome were analysed by Southern blot of canola genomic DNA probed with IPG-1 cDNA (Fig. 7). The results showed that IPG-1 has about 2–5 copies in the canola genome, which means that the canola genome may have a C2 domain gene family. To test whether other members in the gene family interact with sspg1d, an additional two genes in the gene family were isolated by RT-PCR and designated as BnC2d1 and BnC2d2. BLAST analysis indicated that the two genes also contain the C2 domain. Sequence comparison of the two genes with IPG-1 revealed that their identity is as high as 95% in amino acids (Fig. 8). The coding sequences of BnC2d1 and BnC2d2 were individually fused into the GAL4 activating domain of the pGADT7 vector. Yeast mating was used to test the interaction between sspg1d and the two genes. To our surprise, the two genes did not interact with sspg1d, although the experiment was replicated several times. This means that the two IPG-1 homologues may have evolved functions different from IPG-1. This may explain why only one gene that interacts with sspg1d in the canola cDNA library was isolated by yeast two-hybrid screening.

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