Mechanism Of Chinese Wheat Mosaic Virus CRP Phosphorylation Promoting Viral Infection

Successful pathogen infection in plant depends on a proper interaction between the invading pathogen and its host.Post-translational modification（PTM）plays critical role（s）in plant-pathogen interaction.However,how PTM of viral protein regulates plant immunity remains poorly understood.In this study,we found that S162 and S165 of Chinese wheat mosaic virus（CWMV）cysteine-rich protein（CRP）are phosphorylated by serine/threonine-protein kinase SAPK7 in wheat（Ta SAPK7）.Mutational analyses indicated that phosphorylation of CRP at S162 and/or S165 promote CWMV infection in plant.Further study found that NbSRK,the homologue of the kinase Ta SAPK7 in N.benthamiana,interact with CRP.Silencing of NbSRK expression in N.benthamiana affected the phosphorylation of CRP at S162 and S165,inhibited CWMV infection,while transient overexpression of NbSRK promoted CWMV infection.Our results indicated that the kinase NbSRK promote CWMV infection through phosphorylating S162 and S165 of CRP.In order to clarify the molecular mechanism of how S162 and S165 of CRP phosphorylation promotes CWMV infection,we screened a wheat c DNA library through YTH using CRP^S162/165D（phosphorylation-mimic mutant of CRP）as the bait.The result showed that the CRP^S162/165D but not CRP^S162/165A（non-phosphorylatable mutant of CRP）interacts with RNA-binding protein UBP1-associated protein 2C（TaUBA2C）.In addition,silencing of TaUBA2C expression in wheat plants enhanced CWMV infection.In contrast,overexpression of TaUBA2C in wheat plants inhibited CWMV infection,indicating that TaUBA2C is regulator of CWMV infection.The function of TaUBA2C in CWMV infection was further investigated.Our results showed that TaUBA2C induce cell death and H₂O₂ production.Moreover,TaUBA2C is localized in nuclear speckles and is a chromatin-bound protein with RNA-and DNA-binding activities.RIP assays showed that TaUBA2C associates with the pre-m RNAs of TaNPR1,Ta PR1,and Ta RBOHD.Thus,we speculated that TaUBA2C regulates the expression of these three target genes by binding their pre-m RNAs.Based on results above,we concluded that TaUBA2C inhibits CWMV infection through upregulating the expression level of TaNPR1,Ta PR1 and Ta RBOHD to induce cell death and H₂O₂ production.This effect can be supressed by CRP^S162/165D through changing TaUBA2C chromatin-bound status and attenuating it’s the RNA-or DNA-binding activities.Collectively,phosphorylated CRP can bind to TaUBA2C to interfear the formation of TaUBA2C speckles in nuclei to reduce its RNA-and DNA-binding activity,thereby down-regulating the expression of TaNPR1,Ta PR1 and Ta RBOHD,which in turn promotes virus infection.Taken together,our findings provide new knowledge on how CRP phosphorylation affects CWMV infection as well as the arms race between virus and wheat plants.