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

Abstract

The barley stripe mosaic virus (BSMV) triple gene block 1 (TGB1) protein is required for virus cell-to-cell movement. However, little information is available about how these activities are regulated by post-translational modifications. In this study, we showed that the BSMV Xinjiang strain TGB1 (XJTGB1) is phosphorylated in vivo and in vitro by protein kinase CK2 from barley and Nicotiana benthamiana. Liquid chromatography tandem mass spectrometry analysis and in vitro phosphorylation assays demonstrated that Thr-401 is the major phosphorylation site of the XJTGB1 protein, and suggested that a Thr-395 kinase docking site supports Thr-401 phosphorylation. Substitution of Thr-395 with alanine (T395A) only moderately impaired virus cell-to-cell movement and systemic infection. In contrast, the Thr-401 alanine (T401A) virus mutant was unable to systemically infect N. benthamiana but had only minor effects in monocot hosts. 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. However, virus derivatives with single glutamic acid substitutions at Thr-395 and Thr-401 developed nearly normal systemic infections in the monocot hosts but were unable to infect N. benthamiana systemically, and none of the double mutants was able to infect dicot and monocot hosts. 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.

Keywords: Barley stripe mosaic virus; phosphorylation; promotion; protein kinase CK2; triple gene block 1 (TGB1) protein; viral movement..

Fig. 1.

Diagram of _XJBSMV strain infectious clones and cereal and dicot host infectivity test results. (A) Illustration of _XJBSMV infectious clones under the T7 promoter or double cauliflower mosaic virus 35S promoter as described previously (Yuan et al., 2011; Lee et al., 2012). (B) Infectivity assays with in vitro-synthesized gRNAs of barley, wheat, and B. distachyon Bd21. (C) Agroinfiltration was used to initiate infections of N. benthamiana. Typical chlorotic stripes and mosaic symptoms (top) of BSMV appeared on emerging uninoculated leaves by 7–9 dpi. Upper uninoculated leaf tissue was harvested at 12 dpi, and the relative BSMV RNA and CP amounts were evaluated by RT-PCR (middle) and Western blotting with the antibody against BSMV CP (bottom). BSMV RNAγ was detected by RT-PCR with the primer pair BS-10/BS-32 (Supplementary Table S1). (This figure is available in colour at JXB online.)

Fig. 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.)

Fig. 3.

In vitro phosphorylation of _XJTGB1 protein by recombinant CK2 kinase. (A) SDS-PAGE analysis of NbCK2α and HvCK2α purified from E. coli BL21 cells. (B) In vitro phosphorylation of _XJTGB1 protein with the NbCK2α and HvCK2α recombinant proteins and negative controls lacking the kinases. (C) Effects of heparin on in vitro phosphorylation of _XJTGB1 protein. Phosphorylation levels were reduced with increasing amount of heparin. (D) Ability of NbCK2α to use both ATP and GTP as phosphate donors. (E) Divalent metal ion specificity of NbCK2α and the TMV-MP (P30) proteins. The CBB-stained proteins at the bottom of panels (B)–(E) are as indicated as in Fig. 2B. (F) Co-localization of the GFP:_XJTGB1 and DsRed:NbCK2α proteins in N. benthamiana leaf cells. Single localization of GFP:_XJTGB1 and DsRed:NbCK2α proteins are indicated at the top of the panels. Bars, 50 μm. (This figure is available in colour at JXB online.)

Fig. 4.

Thr-401 is the major _XJTGB1 protein site for CK2 phosphorylation. (A) LC-MS/MS analysis of _XJTGB1 protein phosphorylation by NbCK2α. The absence of phosphoric acid (97.9769Da) on the y₁₆ ion fragment demonstrates that Thr-401 is a phosphorylation site for CK2 kinase. (B) Identification of the phosphorylation sites in _XJTGB1 protein mutants by in vitro phosphorylation with HvCK2α and NbCK2α. The radioactive intensities of the _XJTGB1 protein and its phosphorylation mutants indicate the extent of radiolabelling with [γ-³²P]ATP. CBB-stained proteins at the bottom of the panels (B) and (C) are as indicated in Fig. 2B. (C) Phosphorylation comparisons of selected _XJTGB1 protein mutants with wt _XJTGB1 protein to confirm that Thr-401 is the major phosphorylated residue. (This figure is available in colour at JXB online.)

Fig. 5.

Mutants affecting phosphorylation of the _XJTGB1 protein have host-specific effects on systemic infectivity. (A) Symptoms of N. benthamiana elicited after infiltration with an Agrobacterium mixture harbouring pCa-α_XJ, pCa-γ_XJ, and pCa-β_XJ or its phosphorylation site mutants. Upper uninfiltrated leaf tissues were harvested and photographed at 10 dpi (top). CP ELISA (middle) and RNAγ RT-PCR amplification (bottom) were monitored to estimate the infectivity levels. (B, C) Systemic symptoms appearing in barley (B) and wheat (C) after inoculation with pT7-α_XJ and pT7-γ_XJ in vitro transcripts mixed with pT7-β_XJ and various phosphorylation mutant transcripts. Leaves were photographed at 14 dpi (top) and all experiments were repeated three times. (This figure is available in colour at JXB online.)

Fig. 6.

Effects of _XJTGB1 protein phosphorylation on _XJBSMV cell-to-cell movement in N. benthamiana and barley. (A) Fluorescence in N. benthamiana leaves at 3 dpi with an Agrobacterium mixture of pCa-α_ND, pCa-γ_ND:GFP, and pCa-β_XJ or the pCa-β_XJ mutant derivatives. The total bacterial concentrations for infiltration were OD₆₀₀ of 0.08. (B) Fluorescence in barley leaves at 3 dpi with in vitro transcripts of RNAα and RNAγ:GFP plus wt _XJRNAβ or the _XJRNAβ phosphorylation site mutant derivatives. Bars represent 500 μm. (This figure is available in colour at JXB online.)

Fig. 7.

Effect of _XJTGB1 protein phosphorylation mutants on its functions. (A) Comparison of RNA binding by the phosphorylated native _XJTGB1 and double-mutant _XJTGB1_T395A/T401A proteins. (B) GST affinity chromatography comparisons of the _XJTGB1 and double-mutant _XJTGB1_T395A/T401A proteins with the GST:_XJTGB3 protein. The concentrations of the TGB proteins were similar in the experiments, but the _XJTGB1_T395A/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:_XJTGB1_T395A/T401A mutant derivatives and the pGD-TGB2 and RFP:TGB3 plasmids. Bars, 50 μm. (This figure is available in colour at JXB online.)