Abstract
Barley stripe mosaic virus (BSMV) Triple Gene Block1 (TGB1) is a multifunctional movement protein with RNA-binding, ATPase and helicase activities which mainly localizes to the plasmodesmata (PD) in infected cells. Here, we show that TGB1 localizes to the nucleus and the nucleolus, as well as the cytoplasm, and that TGB1 nuclear-cytoplasmic trafficking is required for BSMV cell-to-cell movement. Prediction analyses and laser scanning confocal microscopy (LSCM) experiments verified that TGB1 possesses a nucleolar localization signal (NoLS) (amino acids 95-104) and a nuclear localization signal (NLS) (amino acids 227-238). NoLS mutations reduced BSMV cell-to-cell movement significantly, whereas NLS mutations almost completely abolished movement. Furthermore, neither the NoLS nor NLS mutant viruses could infect Nicotiana benthamiana systemically, although the NoLS mutant virus was able to establish systemic infections of barley. Protein interaction experiments demonstrated that TGB1 interacts directly with the glycine-arginine-rich (GAR) domain of the nucleolar protein fibrillarin (Fib2). Moreover, in BSMV-infected cells, Fib2 accumulation increased by about 60%-70% and co-localized with TGB1 in the plasmodesmata. In addition, BSMV cell-to-cell movement in fib2 knockdown transgenic plants was reduced to less than one-third of that of non-transgenic plants. Fib2 also co-localized with both TGB1 and BSMV RNA, which are the main components of the ribonucleoprotein (RNP) movement complex. Collectively, these results show that TGB1-Fib2 interactions play a direct role in cell-to-cell movement, and we propose that Fib2 is hijacked by BSMV TGB1 to form a BSMV RNP which functions in cell-to-cell movement.