Studies Of The Molecular Interactions Between Maize AKIN?? And P8 Protein Of Rice Black Streaked Dwarf Virus

Maize rough dwarf caused by Rice black streaked dwarf virus(RBSDV)is a widespread and destructive viral disease in China.The sustained control of this disease is difficult because the knowledge on the interaction between RBSDV(Fijivirus,Reoviridae)and maize plants is limited.In this study,we identified the maize ZmAKIN?? protein as an interactor with RBSDV P8,which is the minor core component of RBSDV virion,though a yeast two-hybrid(Y2H)screen by using P8 as the bait.The molecular interaction between RBSDV P8 and ZmAKIN?? and the interplay between ZmAKIN?? and RBSDV was studied.The role of ZmAKIN?? during RBSDV infection and the physiological pathway involved in the interaction between them was explored preliminarily.The interaction between P8 and ZmAKIN??-2 or ZmAKIN??-1 protein in the Nicotiana benthamiana and maize protoplasts were confirmed by BiFC assay.Furthermore,the interaction specificity of P8 and AKIN?? proteins was investigated and the results showed that the interaction was not hosts-specific.We also found that the KIS(kinase interacting sequence)and the first CBS(cystathionine ?-synthase)domains located in the N terminal of ZmAKIN?? protein are essential for the interation with P8 protein.P8 and ZmAKINpy-1/-2 proteins localized to the cytoplasm and nucleus of N.benthamiana and maize protoplasts,and P8 obviously localized to the nucleolus.Interestingly,ZmAKIN??-1/-2 expression influenced the nucleolus localization of P8.In turn,the expression of P8 inhibited the biological activity of ZmAKIN??-1/-2 proteins in snf4? yeast mutant.RBSDV differentially regulated the expression levels of ZmAKIN??-1/-2 genes at different infection stages.In addition,the subcellular localization of ZmAKIN??-1/-2 in maize leaf cells was obviously influenced by RBSDV infection.To understand the roles ZmAKIN??-1/-2 played during RBSDV infection,the ZmAKIN??-1/-2 genes were downregulated by BMV(Brome mosaic virus)VIGS system.The results showed that downregulation of ZmAKIN??-1/-2 expression levels increased the accumulation of RBSDV and did regulate the transcriptional levels of many genes involved in the sucrose and starch metabolism pathways.We further found that the contents of glucose and sucrose were upregulated in ZmAKIN??-1/-2-silenced maize plants.To study the influence of sugar metabolism on the RBSDV infection,the relative expression levels of genes associated with sugar metabolism pathway during RBSDV infection were investigated and the results showed that many of them were regulated.The accumulation of glucose was upregulated and the content of sucrose was downregulated in RBSDV-infected maize plants.Application of glucose to RBSDV-inoculated maize increased the accumulation of virus.Overexpression of N terminal tag-fused ZmAKIN??-2 or the homologues encoded by rice or N.benthamiana could induce the cell death response of N.benthamiana leaves,and the co-expression of P8 did not influence this activity of ZmAKIN??-2.Besides RBSDV,ZmAKIN??-1/-2 could response to distinct virus infections.The transcription levels of ZmAKIN??-1/-2 genes in Sugarcane mosaic virus(SCMV)-or Maize chlorotic mottle virus(MCMV)-infected maize plants were differentially regulated.This study revealed that ZmAKIN?? is a regulator of sugar metabolism,and participated in the interaction between RBSDV and maize plants.Furthermore,the sugar metabolism could be regulated by RBSDV infection.Eventually these results could contribute to the elucidation of the mechanisms underlying the resistance of maize to RBSDV infection and the effective control of maize rough dwarf disease by precisely regulating specific infection-responsive host genes.