| Rice black-streaked dwarf virus(RBSDV)is transmitted by the small brown planthopper(Laodelphax striatellus)in a persistent,propagative manner and cause great yield loss of rice.RBSDV P8 is the core protein of virus particle with a highly conserved NTP domain.It fUnctions as a transcriptional suppressor.However,relative little is known about other biological functions of P8 protein.In this study,we performed yeast two-hybrid(Y2H)screen assay using P8 as bait and obtained an candidate protein,OsSEUSS,for further investigating their roles during RBSDV infection.In addition,we also identified antiviral varieties from indica and japonica rice.The main results were summarized as followed:1.We obtained 10 candidate proteins interacting with P8 by Y2H screen.Among them,the OsSEUSS was further studied.2.Y2H assays showed P8 was interacted with the full-length OsSEUSS,and the NTP domain of P8 and the C-terminal of OsSEUSS were responsive for their interaction,respectively.Bimolecular fluorescence complementation assay revealed that the interaction between P8 and OsSEUSS was in the nucleus of N.benthamiana epidermal cells.Forster resonance energy transfer measured by fluorescence lifetime microscopy and co-immunoprecipitation experiments further comfirmed the interaction between P8 and OsSEUSS in vivo.In addition,P8 did not interact with OsSEUSS homologues,OsSLKl and OsSLK2.3.The expression of OsSEUSS in RBSDV-infected leaves was obviously decreased relative to that in uninfected controls,indicating that OsSEUSS played important roles in RBSDV infection.We generated OsSEUSS over-expression transgenic rice(OE-OsSEUSS)and cas9 mutant(cas-osseuss).Compared with the wide-type ZH11,the primary root length and number of root hairs obviously increased in OE-OsSEUSS but decreased in cas-osseuss plants.Virus inoculation experiment showed that OE-OsSEUSS lines infected by RBSDV were more susceptible,while cas-osseuss lines infected by RBSDV were more resistant,compared to RBSDV-infected ZH11.These results together suggested that OsSEUSS played negative roles in rice defense against RBSDV infection.4.We assessed the sensitivity of seedlings of 20 local rice varieties in Zhejiang province to RBSDV infection by observing symptoms,measuring plant height and counting incidence after artificial inoculation assay.The results showed that the expression of RBSDV mRNA decreased dramatically iN indica rice,such as Shenliangyou 5814 and Y-liangyou 302,compared with that in japonica rice,such as Huai 5 and Xiushui 14.Moreover,the expression of OsMYC2 and OsNPRl in RBSDV-infected indica rice were significantly up-regulated than that in mocked indica plants.These results indicated that the resistance of the indica rice to RBSDV infection might be partly due to activate the jasmonic acid(JA)and salicylic acid(SA)pathway.In conclusion,we demonstrated P8 interacted with OsSEUSS.OsSEUSS negatively regulated rice anti-viral defense.In addition,our data showed that some indica rice varieties with higher expression levels of JA and SA relative genes were more resistant to RBSDV infection,compared with some japonica rice varieties with lower expression levels of JA and SA relative genes.Thus,these results provided a new strategy for screening the resistance of rice varieties to RBSDV. |
