The Research Of Chinese Wheat Mosaic Virus Coat Protein Phosphorylation And Its Functional Mechanism

Chinese wheat mosaic virus(CWMV),causing wheat mosaic virus disease,was a new soil-borne virus identified in Shandong winter wheat in the 1990 s.The wheat plants infected by CWMV generally exhibit yellow mosaic symptoms in leaves,however pathogenic mechanism of CWMV is still unclear.In this study,the overexpression transgenic tobacco(OECP)of CWMV CP gene was obtained by Agrobacterium-mediated transformation assay.The function and mechanism of CP protein in virus infection were analyzed by subcellular localization,protein modification analysis,yeast two-hybrid system and virus-induced gene silencing(VIGS)assays.The specific research results are as follows:1?Four positive OECP generations were obtained,which showed obvious mosaic and dwarfing.In addition,by detecting the photosynthetic rate of transgenic plants,it was discovered that overexpression of CP protein gene could result in significant decrease in the net photosynthetic rate of tobacco plants,indicating that CP protein inhibit the photosynthetic pathway of host plants.Meanwhile,the transcript level of MP gene of CWMV was significantly decreased in OECP infected with CWMV.This result indicated that CP gene could improve the host disease resistance.2?Subcellular localization and immune colloidal gold experiments showed that the CP protein was specifically localized in the chloroplast of the host,and the N-terminal 1-87 amino acids of the CP protein played a decisive role in the localization of the CP protein.However,bioinformatics predicts that CP proteins do not contain chloroplast signal peptides.Thus,the protein mass spectrometry analysis was performed,the results of which showed that the N-terminal of CP protein contains two phosphorylation sites,which are the 45 and 55-position serine(S45,S55).After an analysis of subcellular localization of this two mutated serine,it showed that the signal of CP protein could not be localized in Chloroplast.Our result indicated that S45 and S55 at the N-terminus of CP protein are playing a decisive role in the subcellular localization of CP protein.3?While CP protein was used as bait,a chloroplast-associated protein NbPSID2 was screened out in tobacco cDNA library.In addition,the interaction between CP protein and NbPSID2 was confirmed by yeast two-hybrid system.Silencing NbPSID2 in tobacco by VIGS assay resulted in chlorosis phenotype,suggesting that NbPSID2 is involved in the photosynthesis of tobacco plants.In summary,our results laid the foundation for the cultivation of wheat disease-resistant materials and systematic analysis of the mechanism of CP protein during the CWMV infection.