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Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots.

Hu Y, Li Z, Yuan C, Jin X, Yan L, Zhao X, Zhang Y, Jackson AO, Wang X, Han C, Yu J, Li D - J. Exp. Bot. (2015)

Bottom Line: Substitution of Thr-395 or Thr-401 with aspartic acid interfered with monocot and dicot cell-to-cell movement and the plants failed to develop systemic infections.The mutant XJTGB1T395A/T401A weakened in vitro interactions between XJTGB1 and XJTGB3 proteins but had little effect on XJTGB1 RNA-binding ability.Taken together, our results support a critical role of CK2 phosphorylation in the movement of BSMV in monocots and dicots, and provide new insights into the roles of phosphorylation in TGB protein functions.

View Article: PubMed Central - PubMed

Affiliation: State Key laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China.

No MeSH data available.


Related in: MedlinePlus

Effect of XJTGB1 protein phosphorylation mutants on its functions. (A) Comparison of RNA binding by the phosphorylated native XJTGB1 and double-mutant XJTGB1T395A/T401A proteins. (B) GST affinity chromatography comparisons of the XJTGB1 and double-mutant XJTGB1T395A/T401A proteins with the GST:XJTGB3 protein. The concentrations of the TGB proteins were similar in the experiments, but the XJTGB1T395A/T401A protein had approximate 40% TGB3 protein-binding efficiency compared with the wt XJTGB1 protein. The illustrated binding result is typical of three independent experiments. (C) Co-localization of TGB proteins. Confocal laser-scanning microscopy observation of N. benthamiana leaf epidermal cells co-infiltrated with mixtures of Agrobacterium harbouring GFP:XJTGB1 or the GFP:XJTGB1T395A/T401A mutant derivatives and the pGD-TGB2 and RFP:TGB3 plasmids. Bars, 50 μm. (This figure is available in colour at JXB online.)
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Figure 7: Effect of XJTGB1 protein phosphorylation mutants on its functions. (A) Comparison of RNA binding by the phosphorylated native XJTGB1 and double-mutant XJTGB1T395A/T401A proteins. (B) GST affinity chromatography comparisons of the XJTGB1 and double-mutant XJTGB1T395A/T401A proteins with the GST:XJTGB3 protein. The concentrations of the TGB proteins were similar in the experiments, but the XJTGB1T395A/T401A protein had approximate 40% TGB3 protein-binding efficiency compared with the wt XJTGB1 protein. The illustrated binding result is typical of three independent experiments. (C) Co-localization of TGB proteins. Confocal laser-scanning microscopy observation of N. benthamiana leaf epidermal cells co-infiltrated with mixtures of Agrobacterium harbouring GFP:XJTGB1 or the GFP:XJTGB1T395A/T401A mutant derivatives and the pGD-TGB2 and RFP:TGB3 plasmids. Bars, 50 μm. (This figure is available in colour at JXB online.)

Mentions: To determine whether the RNA-binding affinity of the XJTGB1 protein changed upon phosphorylation, we first used purified wt XJTGB1 and the double mutant XJTGB1T395A/T401A proteins in EMSA with DIG-labelled RNA transcripts (Fig. 7A). The results clearly showed that both proteins bound almost all of the available RNA at 250ng, indicating that the mutant protein did not affect RNA-binding activities (Fig. 7A, panels 1 and 2). Next, to determine phosphorylation effects directly, the XJTGB1 protein was incubated in phosphorylation assay buffer containing unlabelled ATP, with and without the addition of purified NbCK2α, and EMSA assays were performed to compare the abilities of the non-phosphorylated and phosphorylated XJTGB1 proteins to bind the labelled RNA transcripts (Fig. 7A, panels 3 and 4). In addition, the RNA-binding activities of the phosphorylated wt XJTGB1 and mutant XJTGB1T395A/T401A proteins were compared, but the XJTGB1T395A/T401A protein was found to retain almost the same level of RNA binding as the wt XJTGB1 protein (Fig. 7A, panels 5 and 6). Hence, phosphorylation appeared to have little, if any, effect on the RNA-binding activities of the XJTGB1 protein.


Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots.

Hu Y, Li Z, Yuan C, Jin X, Yan L, Zhao X, Zhang Y, Jackson AO, Wang X, Han C, Yu J, Li D - J. Exp. Bot. (2015)

Effect of XJTGB1 protein phosphorylation mutants on its functions. (A) Comparison of RNA binding by the phosphorylated native XJTGB1 and double-mutant XJTGB1T395A/T401A proteins. (B) GST affinity chromatography comparisons of the XJTGB1 and double-mutant XJTGB1T395A/T401A proteins with the GST:XJTGB3 protein. The concentrations of the TGB proteins were similar in the experiments, but the XJTGB1T395A/T401A protein had approximate 40% TGB3 protein-binding efficiency compared with the wt XJTGB1 protein. The illustrated binding result is typical of three independent experiments. (C) Co-localization of TGB proteins. Confocal laser-scanning microscopy observation of N. benthamiana leaf epidermal cells co-infiltrated with mixtures of Agrobacterium harbouring GFP:XJTGB1 or the GFP:XJTGB1T395A/T401A mutant derivatives and the pGD-TGB2 and RFP:TGB3 plasmids. Bars, 50 μm. (This figure is available in colour at JXB online.)
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Related In: Results  -  Collection

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Figure 7: Effect of XJTGB1 protein phosphorylation mutants on its functions. (A) Comparison of RNA binding by the phosphorylated native XJTGB1 and double-mutant XJTGB1T395A/T401A proteins. (B) GST affinity chromatography comparisons of the XJTGB1 and double-mutant XJTGB1T395A/T401A proteins with the GST:XJTGB3 protein. The concentrations of the TGB proteins were similar in the experiments, but the XJTGB1T395A/T401A protein had approximate 40% TGB3 protein-binding efficiency compared with the wt XJTGB1 protein. The illustrated binding result is typical of three independent experiments. (C) Co-localization of TGB proteins. Confocal laser-scanning microscopy observation of N. benthamiana leaf epidermal cells co-infiltrated with mixtures of Agrobacterium harbouring GFP:XJTGB1 or the GFP:XJTGB1T395A/T401A mutant derivatives and the pGD-TGB2 and RFP:TGB3 plasmids. Bars, 50 μm. (This figure is available in colour at JXB online.)
Mentions: To determine whether the RNA-binding affinity of the XJTGB1 protein changed upon phosphorylation, we first used purified wt XJTGB1 and the double mutant XJTGB1T395A/T401A proteins in EMSA with DIG-labelled RNA transcripts (Fig. 7A). The results clearly showed that both proteins bound almost all of the available RNA at 250ng, indicating that the mutant protein did not affect RNA-binding activities (Fig. 7A, panels 1 and 2). Next, to determine phosphorylation effects directly, the XJTGB1 protein was incubated in phosphorylation assay buffer containing unlabelled ATP, with and without the addition of purified NbCK2α, and EMSA assays were performed to compare the abilities of the non-phosphorylated and phosphorylated XJTGB1 proteins to bind the labelled RNA transcripts (Fig. 7A, panels 3 and 4). In addition, the RNA-binding activities of the phosphorylated wt XJTGB1 and mutant XJTGB1T395A/T401A proteins were compared, but the XJTGB1T395A/T401A protein was found to retain almost the same level of RNA binding as the wt XJTGB1 protein (Fig. 7A, panels 5 and 6). Hence, phosphorylation appeared to have little, if any, effect on the RNA-binding activities of the XJTGB1 protein.

Bottom Line: Substitution of Thr-395 or Thr-401 with aspartic acid interfered with monocot and dicot cell-to-cell movement and the plants failed to develop systemic infections.The mutant XJTGB1T395A/T401A weakened in vitro interactions between XJTGB1 and XJTGB3 proteins but had little effect on XJTGB1 RNA-binding ability.Taken together, our results support a critical role of CK2 phosphorylation in the movement of BSMV in monocots and dicots, and provide new insights into the roles of phosphorylation in TGB protein functions.

View Article: PubMed Central - PubMed

Affiliation: State Key laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China.

No MeSH data available.


Related in: MedlinePlus