Function Analysis Of Barley Stripe Mosaic Virus ?b Protein In Phosphorylation Modification And Interaction With Host Protein Kinase STY46

Phosphorylation is a common post-translational modification that has multiple functions during plant virus infection.Protein kinases regulate virus infectivity and pathogenicity by phosphorylating viral replicase,movement protein,coat protein,and viral suppressor of RNA silencing(VSR).The ?b protein encoded by Barley strip mosaic virus(BSMV)that is a positive single-stranded RNA virus has multiple functions in the battle between plant defense and virus counter-defense,but it is unclear whether ?b protein has phosphorylation modification and what is its effect on virus infection and host response.In this study,the phosphorylation of yb protein and its biological function were analyzed,while an attempt was made to know the mechanism of how host kinase STY46 regulated BSMV infection by interacting with and phosphorylating ?b protein.BSMV yb protein phosphorylation during virus infection was detected by anti-phosphoserine antibody.In vitro phosphorylation assay and LC-MS/MS analysis demonstrated that yb protein was phosphorylated by PKA-like kinase in N.bentlhaniana,and Ser-96 is a major phosphorylation site.Plant cell death response and decreased viral accumulation were induced by the non-phosphorylated mutant virus(BSMVS96A and BSMVS96R)in both dicot and monocot plants,in contrast to that mimic phosphorylation mutant virus BSMVs96D has the same pathogenicity as wild type virus,indicating that phosphorylation status of ?b influenced the pathogenicity and accumulation of BSMV.In further,results showed that VSR activity of yb was significantly attenuated by the non-phosphorylated mutant S96A,whereas that was comparable to wild type yb protein when the mimic phosphorylation mutant S96D was applied.Also,it was found that phosphorylation of yb protein did not affect its self-interaction and multimerization,but enhanced its ability to bind to 21 nt siRNA.These results indicate that phosphorylation of yb protein by PKA is essential for its VSR activity to suppress host RNA silencing.Besides suppressing plant RNA silencing pathway,phosphorylation of yb protein by PKA also inhibits host cell death response.In VSR complementary experiments expressed transiently,constitutively with transgenic plants and by BSMV??b virus,all VSRs from other viruses failed to suppress the cell death response induced by BSMVS96A,but only wild type yb protein.These results demonstrated that suppression of cell death response by yb was functionally distinct from its VSR activities.Therefore,phosphorylation of ?b by PKA at Ser-96 is crucial for balance the virus persistent infection and host optimized response.Additionally,we found that BSMV yb protein directly interacted with the protein kinase NbSYT46 of N.benthamiana in vitro and in vivo,in which the BM domain(aa 19-47)of yb and the conserved amino acid Thr-436 of NbSTY46 kinase were crucial for their interaction with each other.Contrastly,the NbSYT46T436A mutant not only lost its autophosphorylation activity,but also could not interact with or phosphorylate yb protein.BSMV infection did not affect NbSTY46 expression levels and subcellular localization.Up-regulation of NbSYT46,by transient expression or constitutive expression in transgenic plants,could inhibit BSMV infection and replication,whereas down-regulation of NbSYT46 expression would aggravate the symptoms of BSMV and increase viral accumulation in N.benthamiana.Further experiments proved that,depending on its kinase activity,NbSTY46 negatively regulated BSMV infection,but not affected the VSR activity of yb.The competitive BiFC results suggested that NbSTY46 might inhibit the interaction between ?b and replicase ?a of BSMV.Therefore,it was concluded that protein kinase NbSYT46 of N.benthamiana negatively regulates BSMV replication and infection by directly interacting with and phosphorylating yb protein.These results provide new evidence for understanding how viral protein phosphorylation works to keep the balance between the virus persistent infection and host modest response,and the function of plant protein kinase in the virus-host interaction during defense.