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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

Phosphorylation of the XJTGB1 protein in vitro and in vivo. (A) Coomasie Brilliant Blue (CBB) staining of recombinant XJTGB1 protein purified from E. coli cells. Molecular weight markers (Fermentas) are indicated on the left side of the gel. (B) In vitro phosphorylation of purified XJTGB1 protein by cellular kinases present in healthy N. benthamiana extracts in the absence or presence of [γ-32P]ATP or [γ-32P]GTP. After the phosphorylation reactions, the TGB1 proteins were separated by 12.5% SDS-PAGE and the incorporated radioactivity was analysed by autoradiography. Reaction mixtures lacking XJTGB1 protein or N. benthamiana protein extracts served as negative controls. The CBB staining in the lower panel indicates that similar amounts of the XJTGB1 protein were present in each in vitro phosphorylation reaction. (C) In vivo phosphorylation of XJTGB1 protein in N. benthamiana by Western blotting with α-TGB1 polyclonal antibodies and α-threonine antibodies. A mock agroinfiltration lacking XJRNAβ was used as a negative control and molecular weight markers (Thermo Scientific) were used to estimate the size of the XJTGB1 protein. (D) In vivo phosphorylation of XJTGB1 protein immunoprecipitated (IP) from N. benthamiana was analysed as in Fig. 2C. (This figure is available in colour at JXB online.)
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Figure 2: Phosphorylation of the XJTGB1 protein in vitro and in vivo. (A) Coomasie Brilliant Blue (CBB) staining of recombinant XJTGB1 protein purified from E. coli cells. Molecular weight markers (Fermentas) are indicated on the left side of the gel. (B) In vitro phosphorylation of purified XJTGB1 protein by cellular kinases present in healthy N. benthamiana extracts in the absence or presence of [γ-32P]ATP or [γ-32P]GTP. After the phosphorylation reactions, the TGB1 proteins were separated by 12.5% SDS-PAGE and the incorporated radioactivity was analysed by autoradiography. Reaction mixtures lacking XJTGB1 protein or N. benthamiana protein extracts served as negative controls. The CBB staining in the lower panel indicates that similar amounts of the XJTGB1 protein were present in each in vitro phosphorylation reaction. (C) In vivo phosphorylation of XJTGB1 protein in N. benthamiana by Western blotting with α-TGB1 polyclonal antibodies and α-threonine antibodies. A mock agroinfiltration lacking XJRNAβ was used as a negative control and molecular weight markers (Thermo Scientific) were used to estimate the size of the XJTGB1 protein. (D) In vivo phosphorylation of XJTGB1 protein immunoprecipitated (IP) from N. benthamiana was analysed as in Fig. 2C. (This figure is available in colour at JXB online.)

Mentions: In order to explore phosphorylation in vitro, the full-length XJTGB1 protein was expressed as a C-terminal His-tagged fusion protein and purified to near homogeneity by Ni-affinity chromatography (Fig. 2A). A soluble protein kinase is known to be present in tobacco species (Hung et al., 2014), and hence the purified XJTGB1 protein was first assayed for phosphorylation using N. benthamiana protein extracts as a kinase source. The first control reaction containing [γ-32P]ATP and cytoplasmic extracts without the XJTGB1 protein resulted in no distinct labelled products (Fig. 2B, lane 1). The corresponding control with the XJTGB1 protein and [γ-32P]ATP alone suggested that the XJTGB1 protein was not autophosphorylated in vitro (Fig. 2B, lane 2). In contrast, when both the cytoplasmic extracts and the XJTGB1 protein were present, a radioactive phosphorylated product co-migrated with the XJTGB1 protein (Fig. 2B, lane 3). These results provide evidence that the XJTGB1 protein is phosphorylated in vitro with [γ-32P]ATP by a soluble kinase in the N. benthamiana extracts. The vast majority of protein kinases use ATP as an exclusive phosphate donor, whereas CK2 can effectively use either ATP or GTP (Matsushita et al., 2000); hence we carried out phosphorylation comparisons with GTP to obtain clues about the identity of the kinase involved in XJTGB1 phosphorylation. Autoradiography of the phosphorylated products revealed a single intense radiolabelled band when either [γ-32P]ATP or [γ-32P]GTP was used as a phosphoryl donor (Fig. 2B, lanes 3 and 4), implying that XJTGB1 is phosphorylated by a CK2-like kinase in N. benthamiana.


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)

Phosphorylation of the XJTGB1 protein in vitro and in vivo. (A) Coomasie Brilliant Blue (CBB) staining of recombinant XJTGB1 protein purified from E. coli cells. Molecular weight markers (Fermentas) are indicated on the left side of the gel. (B) In vitro phosphorylation of purified XJTGB1 protein by cellular kinases present in healthy N. benthamiana extracts in the absence or presence of [γ-32P]ATP or [γ-32P]GTP. After the phosphorylation reactions, the TGB1 proteins were separated by 12.5% SDS-PAGE and the incorporated radioactivity was analysed by autoradiography. Reaction mixtures lacking XJTGB1 protein or N. benthamiana protein extracts served as negative controls. The CBB staining in the lower panel indicates that similar amounts of the XJTGB1 protein were present in each in vitro phosphorylation reaction. (C) In vivo phosphorylation of XJTGB1 protein in N. benthamiana by Western blotting with α-TGB1 polyclonal antibodies and α-threonine antibodies. A mock agroinfiltration lacking XJRNAβ was used as a negative control and molecular weight markers (Thermo Scientific) were used to estimate the size of the XJTGB1 protein. (D) In vivo phosphorylation of XJTGB1 protein immunoprecipitated (IP) from N. benthamiana was analysed as in Fig. 2C. (This figure is available in colour at JXB online.)
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Figure 2: Phosphorylation of the XJTGB1 protein in vitro and in vivo. (A) Coomasie Brilliant Blue (CBB) staining of recombinant XJTGB1 protein purified from E. coli cells. Molecular weight markers (Fermentas) are indicated on the left side of the gel. (B) In vitro phosphorylation of purified XJTGB1 protein by cellular kinases present in healthy N. benthamiana extracts in the absence or presence of [γ-32P]ATP or [γ-32P]GTP. After the phosphorylation reactions, the TGB1 proteins were separated by 12.5% SDS-PAGE and the incorporated radioactivity was analysed by autoradiography. Reaction mixtures lacking XJTGB1 protein or N. benthamiana protein extracts served as negative controls. The CBB staining in the lower panel indicates that similar amounts of the XJTGB1 protein were present in each in vitro phosphorylation reaction. (C) In vivo phosphorylation of XJTGB1 protein in N. benthamiana by Western blotting with α-TGB1 polyclonal antibodies and α-threonine antibodies. A mock agroinfiltration lacking XJRNAβ was used as a negative control and molecular weight markers (Thermo Scientific) were used to estimate the size of the XJTGB1 protein. (D) In vivo phosphorylation of XJTGB1 protein immunoprecipitated (IP) from N. benthamiana was analysed as in Fig. 2C. (This figure is available in colour at JXB online.)
Mentions: In order to explore phosphorylation in vitro, the full-length XJTGB1 protein was expressed as a C-terminal His-tagged fusion protein and purified to near homogeneity by Ni-affinity chromatography (Fig. 2A). A soluble protein kinase is known to be present in tobacco species (Hung et al., 2014), and hence the purified XJTGB1 protein was first assayed for phosphorylation using N. benthamiana protein extracts as a kinase source. The first control reaction containing [γ-32P]ATP and cytoplasmic extracts without the XJTGB1 protein resulted in no distinct labelled products (Fig. 2B, lane 1). The corresponding control with the XJTGB1 protein and [γ-32P]ATP alone suggested that the XJTGB1 protein was not autophosphorylated in vitro (Fig. 2B, lane 2). In contrast, when both the cytoplasmic extracts and the XJTGB1 protein were present, a radioactive phosphorylated product co-migrated with the XJTGB1 protein (Fig. 2B, lane 3). These results provide evidence that the XJTGB1 protein is phosphorylated in vitro with [γ-32P]ATP by a soluble kinase in the N. benthamiana extracts. The vast majority of protein kinases use ATP as an exclusive phosphate donor, whereas CK2 can effectively use either ATP or GTP (Matsushita et al., 2000); hence we carried out phosphorylation comparisons with GTP to obtain clues about the identity of the kinase involved in XJTGB1 phosphorylation. Autoradiography of the phosphorylated products revealed a single intense radiolabelled band when either [γ-32P]ATP or [γ-32P]GTP was used as a phosphoryl donor (Fig. 2B, lanes 3 and 4), implying that XJTGB1 is phosphorylated by a CK2-like kinase in N. benthamiana.

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