Function Analysis Of Suppressor Of RNA Silencing Encoded By Strawberry Vein Banding Virus

In plant,RNA silencing plays a important role in antiviral defense.To counteract host defense,plant viruses encode viral suppressors of RNA silencing(RSV)that target distinct effector molecules in the RNA silencing pathway.The Strawberry vein banding virus(SVBV)gene P6 encodes a multifunctional protein involving in trafficking of viral RNA,determining of host symptoms,suppressing RNA silencing and forming of inclusion bodies.In this study,we show that P6 protein encoded by SVBV is a unique RNA silencing suppressor as determined by co-infiltration assays in Nicotiana benthamiana line 16 c.P6 protein suppresses GFP silencing induced by single-strand RNA,not by double-strand RNA.The deletion mutagenesis assays indicate that the nuclear localization signal of P6 effects silencing efficiency in N.benthamiana line 16 c.Furthermore,we further research results suggest that P6 protein not only cannot recovery endogenous PDS gene systemic silencing triggered by Tobacco rattle virus but cannot suppress endogenous PDS/Su gene systemic silencing.1.P6 protein is a unique RNA silencing suppressor by Strawberry vein banding virus(SVBV).To investigate which proteins encoded by SVBV participated in suppression of RNA silencing,we employed a transient silencing suppression assay based on the GFP transgenic Nicotiana benthamiana line 16 c and only P6 protein was found to suppress GFP local silencing.GFP fluorescence could be observed at 6dpi in the N.benthamiana line 16 c leaves co-infiltrated with the Agrobacterium tumefaciens harboring the GFP gene and the P6 gene,between GFP mRNA and GFP protein accumulation were increased significantly by Northern blot and Western blot,but GFP siRNAs were reduced remarkably by RNA blot.These results indicate that P6 protein is a unique RNA silencing suppressor by Strawberry vein banding virus(SVBV).2.P6 suppresses GFP silencing induced by single-strand RNA,not by double-strand RNA.In plants,PTGS can be triggered by sense RNA(S-PTGS)or inverted repeat RNA(IR-PTGS).To determine the key steps targeted by P6 in RNA silencing pathway,we examined their abilities to suppress S-PTGS and IR-PTGS.For S-PTGS and IR-PTGS systems,35S:FP and 35S:dsFP were served as RNA silencing inducers,respectively.We employed A.tumefaciens cultures co-infiltrated wild type N.benthamiana,and indicated that P6 suppresses GFP silencing induced by single-strand RNA,not by double-strand RNA.Our results suggest that P6 suppresses ssRNA-triggered RNA silencing and inhibits the generation of siRNAs.3.The nuclear localization signal of P6 effects silencing efficiency in N.benthamiana line 16 c.To explore which domain within the P6 protein is critical for suppressing RNA silencing,we constructed four P6 deletion mutants based on the bioinformaticspredicted P6 secondary structure,containing pCAM-P616 m,pCAM-P6?144-188 aa,pCAM-P6?193-223 aa and pCAM-P6?402-426 aa,respectively.We used A.tumefaciens cultures co-infiltrated wild type N.benthamiana line 16 c,and indicated that P6,P616 m,P6?144-188 aa and P6?193-223 aa all suppress PTGS,but were more weaker than that inhibited by P19.However,the deletion from 402 aa to 426 aa at nuclear localization signal(NLS)was unable to suppress the silencing efficiency.Our results show that the nuclear localization signal of P6 effects silencing efficiency in N.benthamiana line 16 c.4.Transient expressed P6 cannot recovery endogenous PDS gene systemic silencing triggered by Tobacco rattle virus.To elucidate the function of transient expressed P6 protein in virus-induced gene silencing(VIGS)mediated endogenous gene systemic silencing,the endogenous PDS gene was inserted into a modified TRV viral vector(TRV-PDS)to serve as a RNA silencing inducer,and TRV-GFP containing a C-terminal 250 bp fragment of sense GFP RNA was negative control.The PDS-silenced N.benthamiana was infiltrated with PVX-P6 at 12 dpi.Our results suggest that transient expressed P6 cannot recovery endogenous PDS gene systemic silencing triggered by Tobacco rattle virus.5.P6 cannot suppress endogenous PDS gene systemic silencing triggered by Tobacco rattle virus.To further examine the function of P6 protein in the suppression of VIGS-triggered endogenous gene systemic silencing,we tested whether P6 protein could suppress systemic silencing by using distinct endogenous PDS/Su gene based on Tobacco rattle virus(TRV).Besides,producing a transient expressed P6 protein vector estabished into PVX.We employed A.tumefaciens cultures infiltrated wild type N.benthamiana.The N.benthamiana plants infiltrated with PVX–P6 showed severe mosaic,malformation and fresh leaf down-curl in their systemically infected leaves at 10 dpi.P6 transient expressed or mock N.benthamiana plants were infected with TRV-PDS or TRV-GFP and indicated that transient expressed P6 cannot suppress endogenous PDS gene systemic silencing triggered by Tobacco rattle virus at 30 dpi.Furthermore,wild type,transgenic P6 or transgenic TYLLCCV ?C1 N.benthamiana plants were infected with TRV-PDS or TRV-GFP.The results suggest that stable expressed P6 cannot suppress endogenous PDS/Su gene systemic silencing triggered by Tobacco rattle virus at 12 dpi.Taken togather,these results suggest that P6 cannot suppress endogenous PDS gene systemic silencing.