A Role Of Autophagy In The Transmission Of Rice Gall Dwarf Virus By Recilia Dorsalis

Rice gall dwarf virus(RGDV)is mainly transmitted by leafhopper vector Recilia dorsalis.During recent years,the research on the interaction between R.dorsalis and RGDV has been increasing.However,little has been known about the antiviral mechanism of R.dorsalis innate immune system.Viruses can invade host cells to achieve its survival and proliferation.Autophagy,as an effective defense,has been demonstrated to protect cells from pathogenic microorganisms infection.Here,we used the system of RGDV and R.dorsalis to investigate the effect of autophagy on viral infection and transmission by insect vectors.This study mainly focused on whether RGDV can induce autophagy in insect vectors to affect viral replication and spread,further revealing a mechanism for the interaction of RGDV with insects.Firstly,the full nucleic acid sequence of the autophagy Marker protein Atg8 gene were identified and amplified from the transcriptome of R.dorsalis with the specific primers designed.The PCR products of Atg8 were integrated to pDEST17 prokaryotic expression vector and then were transformed into Rosetta for protein expression and SDS-PAGE was used to collect the target protein bands.After immunizing the mice with purified protein,the anti-Atg8 polyclonal antibody was prepared and then was crosslinked with fluorescein.Immunofluorescence techniques were used to study the interaction of autophagy protein Atg8 and coat protein of RGDV P8 in the culture cells of R.dorsalis.The results showed that RGDV infection could stimulate autophagy in RGDV cells,which in turn promote the replication and spread of RGDV in the cells.Subsequently,to study the cellular localization of autophagy protein Atg8 and coat protein of RGDV P8,the tissues and organs of R.dorsalis with RGDV dissected were subject to immunofluorescence assay.Interestingly,Autophagy only occurred in the intestine in the pathway of RGDV infection and the specific autophagy protein was not detected in the salivary glands,testis and ovaries.The result of this part indicated that RGDV specifically induced the autophagy in the cells of the intestine but not in other organs of vector.In order to clarify the effects of RGDV-induced cell autophagy on the replication and spread of the virus,Autophagic inhibitor(3-MA),autophagic inducer(Rapamycin)and RNAi technology were applied to inhibit or induce autophagy in both cells and whole body of R.dorsalis.The result of both immunofluorescence techniques and RT-qPCR showed that autophagy had been stimulated during virus infection and was beneficial to the spread of virus in cells or insects body.Meanwhile,inhibition of autophagy stunted the spread of the virus.Experiment on virus transmission furtherly indicated that the induction of autophagy led to an acceleration of the virus transmission.Overall,RGDV could induce autophagy in both culture cells and insects body of R.dorsalis,and induction of autophagy can promote the RGDV replication and the spread of viral cell to cell movement.The results of this study will bring new insight tothe further study of the mechanism of interaction between RGDV and insect vector.