The Molecular Mechanism Of Southern Rice Black-streaked Dwarf Virus Manipulating Host Plants To Attract Transmission Vectors

Southern rice black-streaked dwarf virus(SRBSDV),which belongs to the genus Fijivirus,family Reoviridae,is transmitted by the migratory insect white-backed planthopper(WBPH,Sogatella furcifera)in a persistent,circulative and propagative manner.It is the most severe viral disease affecting rice production in southern China in the past decade.Previous research in our laboratory found that SRBSDV infected plants can attract non-viruliferous WBPH,which is conducive for the non-viruliferous vector to acquire virus from the infected plants and promote the spread of SRBSDV.The research focused on molecular mechanism of the interactions between SRBSDV-Rice-WBPH which will provide a basis for understanding the epidemic and disaster mechanisms and carry our more effective prevention and control of SRBSDV.In this study,the molecular mechanism of how SRBSDV manipulating rice plants to attact non-viruliferous WBPH is analyzed,and the following conclusions are obtained:1.SRBSDV encoded P6 is an effector that enhanced the attractiveness of rice to nonviruliferous WBPH.The “Y” tube olfactory instrument was used to test the preference of non-viruliferous WBPH between transgenic rice plants expressing SRBSDV encoded proteins and wild-type rice plants.The results showed that the rice plants expressing virus P6 protein significantly enhanced the attraction ability to non-viruliferous WBPH,while the attraction ability of transgenic rice expressing other 10 proteins of the virus did not change.2.Ethylene signaling mediates the virus induced attractiveness enhancement of rice to non-viruliferous WBPH.We tested the major defense signals involved in enhancing the attractiveness of WBPH in SRBSDV infected rice by using RNAi mutants.It was shown that by silencing the key gene in ethylene signaling,the SRBSDV infected rice plants no longer had the enhanced attractiveness of planthoppers,while silencing the key gene in jasmonic acid signaling or weakening the salicylic acid signaling,the infected rice plants still maintained stronger attractbility to planthoppers than the healthy plants.3.SRBSDV infection and viral P6 overexpression inhibite the downstream ethylene signaling in rice.The results of RT-q PCR showed that compared with healthy plants,several ethylene responsive genes were significantly down-regulated in infected plants,and P6 overexpression plants have similar effect.Gas chromatographic showed that the ethylene release rate of SRBSDV infected plants decreased significantly.4.Ethylene signaling negatively regulates the attractiveness of rice plants to nonviruliferous WBPH.By exogenous application of ethylene signaling regulator,it was found that ethylene signaling inhibitor treatment could significantly improve the attractiveness of rice to non-viruliferous WBPH,while ethylene synthesis precursor treatment or ethylene directly treatment reduced the attractiveness.Furthermore,ethylene signaling mutant also significantly increase the attractiveness of rice to WBPH while the enhanced ethylene signaling transgenic rice plants had reduced attractiveness.5.SRBSDV encoded P6 protein interacted with OsEIL2 in nucleus,a key transcription factor in rice ethylene signaling.By yeast two-hybrid assay,it was found that SRBSDV encoded P6 interacts with OsEIL2.Subsequently,the interaction was confirmed by Bi FC,GST pull-down,and Split Luciferase assay,and the Bi FC results showed that the interaction occurred in nucleus.6.SRBSDV encoded P6 only interacted with OsEIL2 of the rice EILs family,and the interaction impaired the ethylene responses in the aboveground part of rice plants.Yeast twohybrid assay further confirmed that among the OsEILs transcription factors family,only OsEIL2 interacts with P6.The rice ethylene double response assay showed that the enlongation of the coleoptiles of P6 transgenic rice seedlings was inhibited by ethylene treatment,while the root enlongation was inhibited normally by ethylene treatment.Since OsEIL2 was corresponding to the effect ethylene response in the coleoptiles while OsEIL1 was corresponding to the root,these results indicated that there was a specific interaction between P6 and OsEIL2,and this interaction supressed the ethylene response in aboveground part of rice plants.7.OsEIL2 mediates the enhancement of the attractiveness to WBPH by SRBSDV infection.The oseil2 mutant was generated to test the attractiveness to WBPH.In the oseil2 mutant plants,compared to the healthy ones,SRBSDV infected plants do not have enhanced attractiveness to planthoppers.In addition,compared with wild-type plants,oseil2 mutant had stronger attractiveness to planthoppers,indicating that the loss of OsEIL2 could enhance the attractiveness to non-viruliferous WBPH.8.The subcellular localization of P6 and OsEIL2 was identified,and the interaction between P6 and OsEIL2 was found to reduce the accumulation of OsEIL2 in nucleus.P6 and OsEIL2 were fused with fluorescent protein and transiently expressed in tobacco leaves.Laser confocal microscopy showed that P6 was located in both cytoplasm and nucleus,while OsEIL2 was localized in nucleus only.When P6 and OsEIL2 were co-expressed,the accumulation of OsEIL2 in nucleus was significantly reduced,and nuclear transport inhibitor treatment could impair the reduction.9.The self-interaction domain of OsEIL2 was clarified,and it was found P6 could interfere with the self-interaction of OsEIL2.Yeast two-hybrid assay and Bi FC confirmed that OsEIL2 can self-interact,and the interaction domain is the C-terminus of OsEIL2.When co-express with P6,the self-interaction of OsEIL2 was interfered,indicating that P6 might inhibit ethylene signaling transduction by interfering with the formation of OsEIL2 dimer.In summary,this research focused on the molecular mechanism of how SRBSDV manipulates the rice plants to attract the non-viruliferous WBPH.It was found that by SRBSDV infection,its P6 protein interacted with the key transcription factor OsEIL2 of ethylene signaling,and the interaction interfered with the formation of OsEIL2 dimer and reduced its accumulation in the nucleus,which impaired the ethylene signaling in the aboveground part of rice plants,thereby attracting the non-viruliferous vectors to acquire the virus.These results reveal a new mechanism of virus-host-vector tripartite interaction,providing new ideas for the analysis of the disaster mechanism of SRBSDV,as well as scientific and green prevention and control of the disease.