Studies On The Nuclear Localization Of Maize Chlorotic Mottle Virus Coat Protein And Its Possible Function

In recent years,com lethal necrosis disease(CLND)became one of the important diseases causing severe threat to maize(Zea mays L.)production in several Asian and African countries.Maize chlorotic mottle virus(MCMV)can cause CLND by synergistic infection with one or several other viruses in the Potyviridae family such as Maize dwarf mosaic virus,Wheat streak mosaic virus or Sugarcane mosaic virus.MCMV is the only known member of the genus Machtomovirus in the family Tombusviridae.The genome of MCMV is a positive-sense single-stranded RNA and it produces a 1.47 kb subgenomic RNA 1(sgRNA1)and a 0.34 kb subgenomic RNA2.MCMV CP was produced from sgRNAl and its functions are unknown aside from encapsidation.In this study,we investigated the subcellular localization of MCMV CP and its nuclear importin pathway.The functions of nuclear importin factors ZmIMPala and ZmIMPalb were also explored during MCMV infection,which established the foundation for studying the function of MCMV CP in the nucleus.The subcellular localization of MCMV CP was investigated by transient expression through Agrobacterium infiltration and maize protoplasts transfection.MCMV CP localized in the nucleus especially in the nucleolus in Nicotiana benthamiana epidermal cells,and could be imported into the nucleus in maize protoplasts.Moreover,bimolecular fluorescence complementation(BiFC)suggested that CP dimers/polymers were predominately localized to the nucleus.A series of deletion mutants were constructed and transiently expressed in N.benthamiana epidermal cells to determine the region involved in MCMV CP nuclear localization.The results indicated that the N-terminal basic amino acids-rich region(1-43 aa)was essential for the nuclear localization of MCMV CP.The nuclear import pathway of MCMV CP was investigated.BiFC assays verified that MCMV CP could interact with the receptor factors ZmIMPala and ZmIMPalb,which indicated that MCMV-CP could be imported into the nucleus by the classical importin α/β pathway.Moreover,the N-terminal basic amino acids-rich region(1-43 aa)could mediate the interaction between MCMV CP and ZmIMPα1a and ZmIMPalb,which further implied the importance of 1-43 aa in the nuclear import of MCMV CP.The roles of ZmIMPα1a and ZmIMPalb were investigated during MCMV infection.The relative expression of ZmIMP α1a and ZmIMPα1b was determined in the first systematically infected leaves after MCMV infection.The data showed that the relative expression of ZmIMPα1a and ZmIMPα1b was significantly upregulated at 7 and 10 dpi.Brome mosaic virus(BMV)mediated virus-induced gene silencing system was used to silence the expression of ZmIMPα1a and ZmIMPα1b.Subsequently,BMV-inoculated leaves were challenged with MCMV.It showed that MCMV RNA amounts were decreased in maize plants in which both ZmIMPala and ZmIMPα1b were silenced(BMV-IMPα1/MCMV)compared with the control plants(BMV-GFP/MCMV),which suggested that ZmIMP ala and ZmIMPα1b facilitated MCMV infection.To investigate the function of MCMV CP in the nucleus,the CP was used as a bait to screen the normalized yeast two hybrid maize cDNA library.We found that CP could interact with MCMV P32 which had the transcriptional activation function.The luciferase reporter system was constructed to investigate whether the protein had tanscriptional regulation function.The results revealed that MCMV CP could activate the expression of the reporter gene in the maize protoplast using the luciferase reporter system,which implied that MCMV CP has the transcriptional activation function.