Establishment Of Serological Detection System For Sugarcane Streak Mosaic Virus And Study On The Mechanism Of Nicotiana Benthamiana Cell Necrosis Caused By P1 Protein

Sugarcane is an important sugar and energy crop in China.Affected by Sugarcane mosaic disease,sugarcane yield and quality are declining day by day.sugarcane streak mosaic virus is one of the main pathogens of SMD.At present,SCSMV is mainly detected by electron microscope and molecular biology technology,but there are few reports on serological detection of SCSMV.In order to further enrich the serological detection methods of SCSMV and facilitate the follow-up research,P1 gene was isolated from susceptible sugarcane samples and fused with pET28a prokaryotic expression vector.The recombinant plasmid pET28a-P1SCSMV was transferred into E.coli BL21 strain for induction and purification,and the PI concentrated protein was used to immunize New Zealand white rabbits to obtain SCSMV P1 polyclonal antiserum.In order to verify the specificity and sensitivity of polyclonal antiserum,Western blot detection was carried out on P1 protein concentrate diluted samples and field sugarcane samples,and it was found that polyclonal antiserum could specifically and sensitively detect the target protein,and the polyclonal antiserum was proved to be suitable for SCSMV detection with high specificity and sensitivity by Enzyme linked immunosorbent assay and Dot-blot hybridization.Previous studies have shown that SCSMV P1 is a multifunctional protein with silencing inhibitory activity and strong pathogenicity,and P1 also has nuclear localization signals.Nuclear localization signal mutation will affect its silencing inhibitory activity and pathogenicity.However,it is still unknown how SCSMV P1 protein exerts its biological function and promotes virus infection.P1 nuclear localization signal is very important for its biological function.PVX-P1 and its mutants were heterogeneously expressed on Nicotiana benthamiana,the Northern blot results showed that PVX-P1 promoted the up-regulation of PVX accumulation significantly,while the mutants nlsⅠm,nlsⅡm,nlsm,NLSm and NESm had no significant effect on PVX accumulation.In order to determine whether P1 protein interacts with itself,this study was verified by Yeast two-hybrid system,Bimolecular fluorescent complimentary and Co-immunoprecipitation.The results showed that P1 protein could interact with itself,but the mutation of P1 nuclear localization signal led to its inability to interact with itself.The pathogenic function of P1 protein requires its steady-state distribution in the cytoplasm and nucleus.In order to determine whether the nuclear transport receptor proteins Importin a and Importin β in N.benthamiana affect the pathogenicity of P1 protein,the function of P1 protein was investigated by TRV-induced gene silencing technology.The results of trypan blue staining showed that the necrotic symptoms caused by inoculation of PVX-P1 on effectively silenced NbImportin a and NbImportin βplants were slighter than those of the control,indicating that N.benthamiana nuclear transport receptor protein did affect the pathogenic function of P1 protein.The localization of P1 protein was observed in N.benthamiana cells by transient expression experiment.The results showed that P1 was located in the endoplasmic reticulum and produced aggregated vesicles near the endoplasmic reticulum.The expression of endoplasmic reticulum chaperone gene and unfolded protein response regulated gene in N.benthamiana was analyzed by quantitative real-time PCR.The results showed that P1 protein significantly upregulated UPR-related regulatory genes,while P1 mutants had little effect on related genes,indicating that P1 protein could activate Endoplasmic reticulum stress,and ER chaperone gene NbBLP4 and UPR regulatory gene NbbZIP60 silencing could significantly enhance PVX-P1 infection,indicating that P1 protein pathogenicity was affected by its regulation.In order to verify the specific pathogenicity of P1 protein,semi-quantitative RT-PCR showed that the expression of NbbZIP60S was significantly lower than that of NbbZIP60U,due to PVX-P1 infection,indicating that P1 protein was involved in the regulation of IRE1-bZIP60 pathway in N.benthamiana.We further speculated that P1 protein may bind to bZIP60 mRNA and inhibit its splicing mechanism,so as to promote virus infection.RNA binding protein immunoprecipitation showed that P1 protein could bind to bZIP60 mRNA.In summary,this study has established a complete serological detection system to provide a rapid and effective detection method for the prevention and control of SMD.It further clarified the key role of P1 nuclear localization signal on its own interaction and pathogenic function.In addition,the pathogenicity of P1 protein is affected by the proteins Importin α and Importin β of N.benthamiana.To resist P1 protein infection,N.benthamiana initiates the basic defense response of UPR.On this basis,P1 protein binds to bZIP60 mRNA in IRE1-bZIP60 in the UPR regulatory pathway of N.benthamiana and inhibits its splicing to promote virus infection.As a result,N.benthamiana cannot alleviate the pressure of UPR.When the ER is under long-term stress,the expression of autophagy or apoptosis-related genes is initiated,which will eventually cause complete necrosis of N.benthamiana cells.