Subcellular Localization Maintains The Mechanism Of Action And Pathogenicity Of Sugarcane Streak Mosaic Virus P1 Protein Inhibitor

Sugarcane streak mosaic virus(SCSMV)is one of the three main pathogens causing sugarcane mosaic disease(SMD)on sugarcane.SCSMV belongs to the genus of Poacevirus in the family of Potyviridae.Previous studies showed subcellular localization of suppressor encoded by plant virus could significantly affect RNA silencing suppressor(RSS)activity and regulate viral pathogenicity.The PI protein encoded by SCSMV can suppresse single-stranded RNAs(ssRNAs)induced local RNA silencing.However,whether P1 could inhibit double-stranded RNA induced local RNA silencing,or P1 protein has specific function in plant-virus interactions are obscure.We confirmed that PI protein encoded by SCSMV is a RNA silencing suppressor by Agrobacterium tumefaciens-mediated transient expression in wild-type Nicotiana benthamiana.P1 protein was infiltrated with single strand GFP or ssGFP and dsFP contained hairpin structure in N.benthamiana,the P19 protein encoded by TBSV was used as positive control and empty vector was used as negative control.After 3 days,we found that the infiltrated area of P1 have strong green fluorescence similarly to P19 under UV light,while nearly no green fluorescence appeared in the infiltrated area of empty vector.The results showed that ssRNAs and dsRNAs induced local RNA silencing could be suppressed by P1 protein.Western blot results are consistent with observation,there are more GFP protein in the P19 and P1 infiltrated areas than that in empty vector infiltrated area.In order to determine whether P1 suppresse systemic RNA silencing induced by ssRNAs,co-infiltration of PI with ssGFP were carried out on 16c transgenic N.benthamiana plant.11 days later,83%N.benthamiana plants that expressing PI protein were still appeared strong GFP fluorescence on new emerging leaves,which as well as plants expressing P19 protein.The results demonstrated that P1 could supress systemic RNA silencing.P1 was showed to be a pathogenic determinant by the heterogeneous expression system in this study.PVX-P1 plasmid was constructed by cloning P1 gene into heterologous expression vector PVX.The PVX-GFP used as control.Recombinant virus inoculation was performed by agroinfiltration.At 9 dpi,the PVX-P1 could cause more serious mosaic symptoms and even necrosis regions in leaves than PVX-GFP inoculated plant.All these results showed that PI protein act as a strong pathogenic determinant in virus infection.We firstly found that P1 protein could localized to both cytoplasm and nucleus,but could not entry to nucleolus.Two nuclear localization signals were predicted by bioinformatics software.We got mutants nlslm?nlsllm?nlsIIIm by replace Lysine and Arginine with Alanine.Through statistic the cell numbers of nucleus localization in observed field,we found there are still 35%-40% cells exhibited nucleus localization in single nlsIm or nlsIIm mutant;while less than 10% cells could observed nucleus localization in double nuclear localization signal(NLS)mutant treatment(nlslllm),according to the observation of confocal microscopy.All these results showed the binary NLS are necessary for PI nucleus localization.To explore the relationship between P1 subcellular localization and the RSS acticity,we constructed additional two mutants through adding the SV-40 T-antigene NLS and nuclear export signal(NES)to the N-terminal of the PI protein respectively,which to force PI protein completely entry or out of nucleus.Combination with above NLS mutation,the RSS activity were analysed.The results showed hardly visible fluorescence in the co-infiltrated area of PI mutants with ssGFP after 3 days,which as well as EV and ssGFP infiltrated area.All these results indicate the expressed GFP mRNA were silenced in PI mutants expressed area.All these results demonstrated the distribution ration of cytoplasmic/nucleus was essential for PI RSS activity.To explore the relationship between PI subcellular localization and the pathogenicity of PI protein,all PI mutants were expressed by the PVX-mediated heterogeneous expression system.Recombinant virus was inoculated and the virus induced sypmtoms were observed.The symptom of PVX-P1 exhibited mosaic,stunting,large scale of necrosis on leave and stem after 9 dpi,which were more severe than the PVX-P1 mutants,and the PVX-GFP showed mildest mosaic sympotoms.And the symptom of nlslm and nlsllm are worse than nlslllm,NLSm and NESm.14 days later,the upper leaves of plant infiltrated PVX-P1 occurred completely necrosis,the infiltrated leaves of nlslm or nlsllm became necrotic,but it is lighter than plant infiltrated PVX-P1.DAB staining and trypan blue staining also confirmed that the necrosis caused by P1 was much more serious than others.Western blot and RT-PCR showed that P1 and its mutant can express.Above all,the local RNA silencing and systemic RNA silencing caused by ssRNAs can be suppressed by P1 protein.It can also suppresse local RNA silencing induced by dsRNAs.PI is a strong pathogenic determinant of SCSMV.P1 protein located both in the cytoplasm and nucleus.The changes of subcellular localization would dramatically impair the RSS activity and pathogenicity of P1.