The Mechanism Of Rice Black-Streaked Dwarf Virus Protein P9-1 Regulating The Proliferation Of Virus In Laodelphax Striatellus

Rice virus disease is one of the most serious diseases to rice production,which seriously affects the yield and quality of rice.Rice streaked dwarf virus(RBSDV)and Rice stripe virus(RSV),transmitted by Laodelphax striatellus,are two most harmful viruses to Rice.The interaction between viruses and insects is an important way to analyze virus transmission mechanism and control virus transmission.The non-structural protein P9-1 of RBSDV forms viroplasm during virus infection,which may play an important role in virus replication and infection.In this study,Laodelphax striatellus and rice RBSDV were used as experimental materials.Using P9-1 of RBSDV as bait,the cDNA library of Laodelphax striatellus was screened by yeast two hybridization technology(Y2H),function verification of 26 S proteasome subunit Rpn8 which may be involved in the media transmission was carried out,and the molecular mechanism of Rpn8 was preliminarily analyzed.The main results and conclusions are as follows:(1)P9-1 localized in cytoplasm.the subcellular localization analysis showed that P9-1 was distributed in cytoplasm and existed as a viral inclusion body,which indicated that P9-1 plays an important role in the process of proliferation of viruse.(2)P9-1 interacted with the 26 S proteasome subunit Rpn8 in Laodelphax striatellus.Studying the interactions between virus proteins and insect proteins is the main method to explore the mechanism of virus transmission.Then P9-1 was used as the bait protein to screen the cDNA library of Laodelphax striatellus and finally obtained 11 proteins.Studies have shown that 26 S proteasome is involved in the regulation of virus transmission in Laodelphax striatellus,and Rpn8 is a subunit of the 26 S proteasome.Then,Rpn8 was selected for further analized,and the interaction between Rpn8 and P9-1 was further confirmed in Co-immunoprecipitation(Co-IP)and Bimolecule fluorescence complementary(BiFC)assays.(3)Rpn8 negatively regulated the accumulation of RBSDV in Laodelphax striatellus.To detect whether Rpn8 is involved in the regulation of virus transmission,double-strand RNA of Rpn8(dsRpn8)was synthesized directely in vitro and added into forage to fed the toxic Laodelphax striatellus.And the results showed that the gene encoding coat protein of RBSDV(CP)was up-regulated and the virus accumulation was increased.When Rpn8 RNAi transgenic lines inoculated with RBSDV,higher infected ratio was monitored than that in wild type,indicating that down-regulated expression of Rpn8 was beneficial to the accumulation and transmission of RBSDV in Laodelphax striatellus.(4)RBSDV inhibited the function of 26 S proteasome.The qRT-PCR results showed that the expression of Rpn8 in toxic Laodelphax striatellus was reduced significantly than that in non-toxic Laodelphax striatellus.Further more,the ubiquitinated proteins in toxic Laodelphax striatellus were also higher than that in non-toxic Laodelphax striatellus in Western blot assay.These results indicated that RBSDV might inhibit the function of 26 S proteasome by reducing the expression of Rpn8 in Laodelphax striatellus.(5)Rpn8 interacted with Rpn7.The interaction between the different subunits of proteasome is critical for its function exertion.Yeast double hybridization(Y2H),luciferase complementary assay(LCI)and in vitro immunoprecipitation(pull-down)showed that Rpn8 interacted with Rpn7,another subunit of 26 S proteasome in Laodelphax striatellus,and that P9-1 did not interact with Rpn7.(6)Rpn8 interacted with P2 of RSV.The expression level of Rpn8 in toxic Laodelphax striatellus was lower than that of non-toxic Laodelphax striatellus,indicating that RSV could also affect the activity of 26 S proteasome.Down-regulation of Rpn8 promoted the accumulation of RSV in Laodelphax striatellus,and the increased sensitivity of Rpn8 RNAi transgenic rice to RSV indicated that Rpn8 negatively regulated the accumulation and transmission of RSV in Laodelphax striatellus.And then,we found that Rpn8 interacted with P2 of RSV through Y2 H experiment.Subsequently,the interaction of Rpn8 and P2 was confirmed by LCI and Co-IP,respectively.The above results showed that RSV promoted virus accumulation by interfering with the function of 26 S proteasome in Laodelphax striatellus,and the mechanism was similar to RBSDV.To sum up,RBSDV might regulate the function of 26 S proteasome by reducing the expression of Rpn8,and promoted the accumulation of virus in Laodelphax striatellus.The interaction of 26 S proteasome components is known to determine its function,and this study demonstrated that Rpn8 interacts with Rpn7,and the viral protein P9-1 interacts with Rpn8,but P9-1 did not interact with Rpn7 in Laodelphax striatellus.Therefore,we speculated that RBSDV may affect the interaction between Rpn8 and Rpn7 through P9-1 to inhibit the proteasome function,thus promoting its accumulation in Laodelphax striatellus.