Studies On Interaction Between RSV P3 Protein And A Host Nucleus-localized Protein NbP3IP

Rice stripe virus(RSV)which is transmittd by small brown plant hopper in persistent and circulative-propagative manner,can cause rice stripe disease.RSV as an important viral disease of rice not only infects rice plant,but also can infect wheat,Arabidopsis thaliana and Nicotiana benthamiana.As a negative sense RNA virus,RSV is the type member of the genus Tenuivirus.The genome of RSV contains four single stranded RNAs,which encode seven viral proteins using an ambisense coding strategy.The RNA3 sense strand encodes a size of 23.9 kDa nonstructural protein(p3)that is confirmed to be a RNA silencing suppressor.The p3 protein can prevent single-stranded siRNA and double-stranded siRNA entering to RNA-induced silencing complex(RISC)by recognizing their helical structure and 2’hydroxyl group.The interaction between p3 and host proteins are still largely undiscovered.In our study,one of the candidate colonies designated as N.benthamiana protein interacting with p3(NbP3IP)was screened by yeast two-hybrid assay with RSV p3 as a bait in Nicotiana benthamiana cDNA library.The interaction between p3 and NbP3IP was further verified by Yeast two hybrid(Y2H),Bimolecular Fluorescence complementation(BiFC)and Co-Immunoprecipitation(Co-IP).A nuclear localization signal(NLS)was detected in the amino acide(Klionsky et al)sequence of NbP3IP after bioinformatics analysis and its nuclear-localization was confirmed by confocal observation.The GFP fused NbP3IP was localized as the irregular form in the nucleus with partial nucleolus localization.The ratio of this two localization was about 2:1.After constructing a series of deletion mutants,the nuclear localization signal of NbP3IP was determined at its 31-50 aa.The RNA expression level of NbP3IP was reduced by 40%after RSV infection through qPCR detection.The tobacco rattle virus(TRV)-induced gene silencing(VIGS)system was used to investigate the effect of NbP3IP silencing on RSV infection.The accumulation level of RSV RNAs was higher in NbP3IP-silenced plants than in non-silenced ones.Meanwhile,transgenic Nicotiana benthamiana and rice plants for over-expression of NbP3IP were obtained.After inoculation of RSV,the systemic infection was delayed in transgenic tobacco plants and the viral symptom presented mild compared with wild plants.Northern blot of viral RNA confirmed that RSV accumulated less in NbP3IP over-expressed transgenic plants,which indicated that transgenic plants had more resistant ability to RSV infection.These results suggest that NbP3IP plays an important role in the process of resisting RSV infection.Transient expression of NbP3IP could specifically inhibit the silencing suppressing activity of p3 on GFP transgenic tobacco 16c with no effect on other suppressors like p19,p25 and HC-Pro.The peptide of nuclear localization signal in NbP3IP was indispensable for this inhibitory function to p3.Further investigation explored that NbP3IP could distribute to the peripheral of cellular with the p3 transient expression.On the contrary,transiently expressed NbP3IP could not change the localization of p3,however the intensity of p3 green fluorescence was weakened,indicating that the accumulation of p3-GFP was affected.The protein accumulation of p3 was detected by Western blot,which showed significantly lower level of p3 when it coexpressed with NbP3IP.Two inhibitor chemicals for autophagy(3-MA)and proteasome(MG132)respectively were applied to identify the pathway to degrade p3 when NbP3IP and p3 were expressed together.The results showed that the expression level of p3 coexpressed with NbP3IP was higher in 3-MA treated patch than the one treated with water.However,the accumulation level of p3 was not affected after substituting 3-MA with MG132.Therefore,we suggested that the degradation of p3 protein induced by NbP3IP may depend on the autophagy pathway,rather than the proteasome pathway.The degradation of p3 induced by NbP3IP was inhibited on autophagy-related genes 5 and 7(ATG5 and ATG7)silenced plants,indicating that p3 degradation depends on autophagy pathway.BiFC and CoIP experiments confirmed the interaction between NbP3IP and NbATG8f,indicating that NbP3IP may be involved in the regulation of autophagy pathway.Confocal,TEM and RT-PCR detections revealed that RSV infection could induce autophagy.Silencing of ATG5 or ATG7 promoted RSV infection,while GAPC silencing suppressed RSV infection,indicating that autophagy plays a negative role in the process of RSV infection.In natural host rice of RSV,we cloned a homologous gene OsP3IP,which interacted with OsATG8b and p3,and could also degrade p3 protein through the autophagy pathway.Transgenic overexpression of OsP3IP rice can inhibit RSV infection.In summary,our study identified a nuclear localization protein NbP3IP,interacting with the RSV p3 protein,which could induce p3 degradation via autophagy pathway and consequently inhibit its silencing suppressing activity.Autophagy plays a negative role in RSV infection.This study provides us a new comprehension of complicated relationship between virus and host interaction,viral pathogenicity,and plant antiviral pathway.