Molecular Mechanisms Underlying Cucumber Mosaic Virus Self-regulation During Viral Exclusion From Shoot Apical Meristem

Most plant viruses could not infect host's shoot apical meristem(SAM).Thus,virus-free plants are usually obtained from shoot apices using tissue culture technique in the actual production.It is thought that SAM of plants is virus-free mostly due to RNA silencing,which is usually restrained by viral suppressors of RNA silencing(VSRs)that facilitates viral transient SAM invasion.However,the mechanisms of long-term recovery from transient meristem invasion remain elusive.Cucumber mosaic virus(CMV),infecting more than 1,200 plants species,is one of the most important virus causing serious damages on many commercial crops.The aim of this research was to exploit the molecular mechanisms of the transient SAM invasion of CMV by analysing the relationship between coat protein(CP)and 2b protein.Through analysis of the nuclear and cytoplasm fractions of CMV-infected plants and the localization of GFP-CP fusion proteins,we found that CMV CP was able to target to the nucleus and nucleolus of host cells.Besides,the N-terminal region of CMV CP contains a short arginine(R)-rich region(13RRRRPRR19),which is required for the nucleolus localization of the CMV CP.To explore the function of N terminal R-rich domain,we constructed a mutant virus,CMVNMI,harboring alanine substitutions in the R-rich motif(CPNM1).The infection of CMVNM1 was lower efficient than CMVWT,because of low RNA binding ability and unstable virions induced by mutation in the R-rich motif.Nonetheless,CMVNM1 induces severely curled leaves mainly in the newly emerging tissues and abolishes the apical dominance of infected plants.Distribution of CMVs in the shoot meristem tissues of CMV-inoculated N.benthamiana was examined using in situ hybridization.CMVNM1invaded SAM persistently,whereas CMVwT transiently invaded SAMs and was eliminated from meristem after 14 dpi.Additionally,the coinfection assays of PVX:GFP and CMV demonstrate the same results in the expression and observation of green fluorescent protein in the SAM regions.Those results demonstrate that the mutant of R-rich region in CP enhance the shoot apex regions invasion of CMV,indicating that CPWT negatively regulates virulence of CMV in the SAM of infected N.benthamiana plants.To explore the mechanisms for different infectivity of CMVWT and CMVNM1,we next examined the effects of CPWT and CPNM1 on the 2b-mediated suppression of local GFP silencing using Agrobacterium-medidted co-infiltration assays.The results showed that CMV CPWT efficiently decreased 2b-mediated suppression of local GFP silencing and induced relative more siRNA accumulation compared with CPNMI.Moreover,the attenuation of CMV CP on 2b's suppressor activates was dependent on the high accumulation of CP.The relative ratios of viral siRNAs/gRNA in the CMVWT-infected newly emerging tissues was higher than those of CMVNM1-infected plants,indicating that CMVWT induces more potent amplification of virus-derived siRNAs than CMVNMI in the newly grown tissues.In conclusion,the high concentration of CMV VPWT in the late infection promotes accumulation of viral siRNAs to inhibit viral virulence in the apical meristem.In contrast,CPNM1 induces less potent antiviral silencing in the emerging tissues and persistently invades SAM.In addition,the functions of the nuclear importation of CMV CPWT were analyzed through phosphorylation modification and nucleus-cytoplasmic shuttling assays.The purified CPWT fron Escherichia coli was in vitro phosphorylated in presence of N.benthamiana total protein-kinases extract.According to predicted phosphorylation sites and analysis of mass spectrometry(MS),four phosphorylation sites around the nucleolus localization signals were identified.The alanine substitution mutants of the four phosphorylation sites did not affect virus infection,implying that phosphorylation of CMV CP is not involved in its nuclear localization.When the C terminus of CMV CP was fused with a nuclear localization signal(NLS)or a nuclear export signal(NES),the fused CP would be absent from most nuclei(CPNES)or more abundance in the nuclei(CPN'S).The mutant CMVNLS induced hypersensitive response(HR)in the inoculated leaves and could not systemically infected N.benthamiana plants.The mutant CMVNES possess lower infectivity than CMVWT and the relative ratios of viral siRNAs in the CMVNES-infected upper leaves is higher than those of CMVWT,indicating that CMVNES induces more potent amplification of virus-derived siRNAs than CMVWT.Those results demonstrate that CMV CP's nucleocytoplasmic distribution is of importance for viral infection.Taken together,CMV CPWT negatively regulates the SAM invasion of CMV by attenuating RNA silencing activities of 2b,implying that CMV could control its infectivity balancing antiviral silencing and VSRs-mediated suppression.