The Mechanism On Synthesis Pathway Of Polyamines Medited By Rice Yellow Stunt Virus In Nephotettix Cincticeps Vectors

Rice yellow stunt virus(RYSV),is a negative single stranded RNA virus.It belongs to a species of Nucleorhabdovirus genus in the family of Rhabdoviridae.Its virus particles are bullet-shaped,the only rice nucleorhabdovirus that has been reported.The viruses are mainly transmitted by Nephotettix cincticeps、Nephotettix apicalis、Nephotettix virescens and Nephotettix nigropictus in a persistent-propagative manner.The function of RYSV proteins in plants has been relatively studied by the research team of Dr Rongxiang Fang,but there is still a long way to go in the study of the mechanism about the interactions between the virus and vector insects.In order to further clarify the relationships between RYSV and leafhopper,we used the Yeast two-hybrid system to screen the cDNA library of N.cincticeps by viral M protein as bait.Finally,24 target proteins that may interact with M were captured,which were performed for sequence alignment and functional analysis.The results showed that RYSV M interacted with and the key enzyme of polyamines synthesis pathway in N.cincticeps,ornithine decarboxylase antizyme(OAZ).The co-location system in baculovirus-infected Sf9 cells and the Pull down assay were used to verify interaction relationship between M and OAZ in vitro.The reported results indicate that OAZ can inhibit the synthesis of polyamines in insect cells by degrading polyamines synthase,namely,ornithonine decarboxylase(ODC)with specific binding.In order to define whether RYSV M has a regulation role on polyamines synthesis,we first tested the OAZ and ODC genes expression levels,and HPLC-MS technique was used to detect the synthesis of polyamines at the metabolic level during RYSV infection.The results showed that the transcription and expression of OAZ gene were significantly inhibited after RYSV infection,while those of ODC gene were slightly increased.HPLC-MS showed a significant increase in the content of putrescine in polyamines at the metabolic level during viral infection.Thus,it revealed that RYSV infection decreased OAZ synthesized,which directly led to the increase of ODC.Finally,the synthesis of polyamines was improved.Therefore,we preliminarily speculated that RYSV affected the accumulation of ODC through "hijacking" OAZ by M,thus mediating the level of polyamines synthesis in insect vectors.In order to further understand why RYSV regulates the synthesis of polyamines,we have deeply analyzed the effects of polyamines in RYSY infection in insects.First,knockdown OAZ and ODC expression levels by RNA interference.RT-qPCR and immunofluorescence showed that,after silencing of OAZ gene,the average viral copy number of RYSV in N.cincticeps was increased.Subsequently,the expereiment of feeding putrescine in vitro proved that polyamines could enhance RYSV accumulation significantly.Thus,we believe that polyamines can promote the replication and proliferation of RYSV in insect vectors.In addition,in order to reveal whether increase of.polyamines synthesis during RYSV infection whether effects the vector leafhoppers’growth and development,we detected the transcription and expression of vitellogenin(Vg).The results reveal that,during RYSV infection,both at RNA and protein levels,Vg were up-regulated.Similarly,we have fed putrescine in vitro,to further verify that polyamines was related to the synthesis of Vg.Then we discovered the accumulation of polyamines directly led to the increase of Vg.All these results demonstrate that the accumulation of polyamines during RYSV infection can promote the synthesis of Vg and also affect the development of insects’ ovary and number of their eggs.In summary,our study utilizes some different technologies such as vector cell culture,immunofluorescence,RNAi,RT-qPCR_and Western blotting,etc,to clarify that RYSV could regulate polyamines synthesis pathway.Furthermore,we verify that polyamines could help RYSV accumulate and promote the synthesis of Vg.We reveal a new mechanism for RYSV to use polyamines to complete its own life activities and also reveal a set of interaction mechanisms evolved between viruses and vector insects in the process of long-term evolution.Our study provides new ideas for insect transmission virus research and also provides a theoretical basis for the prevention and treatment of viral diseases.