Functional Analysis Of The Barley Stripe Mosaic Virus TGB1Protein Phosphorylation In Viral Infection And Movement

Barley stripe mosaic virus (BSMV) is the type member of the genus Hordeivirus, and the triple gene block1(TGB1) encoded by RNAβ is a multifunctional58kDa protein with RNA binding, RNA helicase, and ATPase activities. The TGBl protein interacts directly with the TGB3protein and indirectly with the TGB2protein to form heterologous complexes required for co-localization at the plasmodesmata (PD) and BSMV cell-to-cell movement. Our previous studies have indicated that TGB1is the pathogenicity determinant to Brachypodium distachyon. The NDTGB1protein of BSMV ND18strain (NDBSMV) could interact with R gene Bsrl in B. distachyon and elicit the resistance response. However, little information is known about the phosphorylation of BSMV TGB1protein.In order to explore phosphorylation of the TGB1protein, the research on the BSMV Xinjiang isolate (XJBSMV) were carried out. Western blot of the Nicotiana benthamiana leaves infected by xjBSMV with an anti-phospho-threonine (a-pT) antibody, and in vitro phosphorylation assays of the C-terminal His-tagged fusion XJTGB1protein indicated that the XJTGB1protein could be phosphorylated by protein kinase2(CK2) in vivo and in vitro. Through the phosphorylation predictions of the GPS2.1program tool and the Scansite Motif Scanner online server, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and the in vitro phosphorylation assays of XJTGB1and its mutants demonstrated that the major phosphorylation residue on XJTGB1was located at the threonine-401(Thr-401), with a CK2phosphorylation docking site at the threonine-395(Thr-395). The inoculation results of barley, wheat and N. benthamiana by the mutants derived from the xjBSMV infectious clones at the Thr-395and Thr-401sites showed that the infectivity of all the mutants reduced obviously in different degrees compared to the wild type (wt). Through the observation of viral cell-to-cell movement in the inoculated N.benthamiana and barley leaves, it was known that some mutants lost the infectivity due to the limit in the cell-to-cell movement. The data of GST pull-down revealed that phosphorylation regulated the function of TGB1protein via promoting the interaction of the TGB1and TGB3proteins. Therefore, it had an influence on the virus infectivity.Previous studies have found that xjBSMV could infect the maize inbred line B73systemically while NDBSMV could not. In order to identify the pathogenicity determinant, the maize B73were inoculated with the genomic RNA reassortants and the recombinations of NDRNAβ and XJRNAβ. The results demonstrated that the pathogenicity determinant of xjBSMV on B73was the TGB1protein and the key virulence amino acid was the glutamic acid at the404residue (Glu-404). Given the regulatory role of the TGB1phosphorylation in BSMV infection, the NDTGB1protein and its mutants derived from Glu-404and phosphorylated sites were purified. Then phosphorylation of these proteins were compared in the in vitro assays for the mechanism of the pathogenicity determinant in maize. On the other hand, the same site mutants were made at the base of the NDBSMV and xjBSMV infectious clones and the inoculation was carried out on the maize B73. However, it turned out that there was not a strict positive correlation between the phosphorylation levels of the mutant proteins and the infectivity of mutant virus. As a result, it was indicated that the phosphorylation of TGB1protein had involved in the xjBSMV infection in maize while there were other host factors might participate in the process.For further studies of the BSMV host range, some infectious clones of BSMV strains were constructed. Via sap-inoculation or argo-infiltration the maize, millet, sorghum, Nicotiana tobacum, and N. benthamiana were inoculated or infiltrated by BSMV six strains and isolates. The data showed that the BSMV strains and isolates varied a lot among these different hosts. Besides, the infections of BSMV on N. benthamiana were co-regulated by both RNAβ and RNAy. All of the results above provide new references and evidences in the research of BSMV infectivity and pathogenicity mechanism in other hosts.