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.