Competitive hijacking of fibrillarin by TBTV P3 and TVDV CP mediates a complex synergistic interaction

Synergistic interactions among plant viruses often exacerbate disease severity, yet the underlying molecular mechanisms, especially those involved in umbravirus and polerovirus interaction, remain poorly characterized. Here, we investigate the tobacco bushy top disease complex, which is primarily caused by co-infection with tobacco bushy top virus (TBTV, Umbravirus) and tobacco vein distorting virus (TVDV, Polerovirus). Our findings reveal a temporally regulated synergistic relationship: TVDV significantly enhanced TBTV accumulation up to 6 days post-inoculation (dpi), while its own replication was consistently suppressed, with marked inhibition observed at key time points. Correspondingly, co-expression of the two viruses led to elevated levels of the TBTV P3 protein but reduced accumulation of the TVDV coat protein (CP), recapitulating their respective viral accumulation dynamic. Direct interaction between P3 and CP was confirmed through BiFC and co-immunoprecipitation (Co-IP) assays. Further protein interaction assays, including BiFC, Co-IP, and FLIM analyses, demonstrated that both P3 and CP competitively target the nucleolar protein fibrillarin2 (NbFib2), with P3 exhibiting a higher binding affinity. Functional studies demonstrated that NbFib2 is essential for systemic infection, as its silencing markedly attenuated the spread of both viruses. Collectively, our findings demonstrate that competitive hijacking of NbFib2 by TBTV P3 and TVDV CP is essential for virus long-distance movement during co-infection, providing novel mechanistic insights into umbraviruses- poleroviruses synergism.