Exploration Of The Molecular Interaction Between The Capsid Protein Encoded By Rice Black-streaked Dwarf Virus And Small Brown Planthopper Vector

Rice black streaked dwarf disease caused by Rice black streaked dwarf virus(RBSDV),is one of important viral diseases of gramineous crops and causes huge food losses annually.RBSDV belonging to the Fijivirus genus in the family Reoviridae and is known to be transmitted by small brown planthopper(SBPH,Laodelphax striatellus Fallen),in a persistent,circulative and propagative manner in nature.It can not be transmitted by the mechanical inoculation.The viral genome consists of 10 linear double stranded RNAs(S1-S10),in which S10 encodes the viral capsid protein(CP).It is well known that the capsid protein of viruses plays an important role in vector transmission of viruses.Deciphering the function and mechanism of CP involved in the process of RBSDV transmission by L.striatellus is meaningful to uncover the epidemic tendency and establish a scientific system for prevention and control of Rice black streaked dwarf disease.Therefore,the molecular interaction mechanism between RBSDV coat protein and its insect vector L.striatellus was investigated in this study.The results were summarized as follows:1)Screening of vector factors interacting with RBSDV coat proteinIn order to clarify the function of RBSDV CP in virus transmission by insect vector,yeast two-hybrid assay was performed to screen SBPH cDNA library and we identified seventeen protein genes that involved in various biological processes.After full length cloning followed by interaction verification,we finally identified five protein candidates interacting with RBSDV CP.These five interacting proteins include receptor for activated protein kinase C(RACK1),cuticular protein tweedle motif 3(CPT3),procollagen-lysine 1,2-oxoglutarate 5-dioxygenase 3-like(PLOD),and prefoldin subunit 2-like protein and paxillin-like isoform 2.2)Interaction mechanism between RBSDV CP and LsRACKl ofL.striatellusRACK1 is a scaffold protein composed of seven WD40 domains and involved in a variety of signaling transduction.The directly interaction between RBSDV CP and LsRACK1 were verified by Co-IP and GST Pull-down assays in vitro.Coat protein mutants were constructed into yeast two-hybrid vectors according to the transmembrane domain prediction in an online website(http://www.cbs.dtu.dk/service-s/TMHMM/).The analysis results showed that the N-terminal 1-270 amino acids of CP(muCP-N)and C-terminal 268-315 amino acids of LsRACK1 were the smallest interaction domains.qRT-PCR was performed to analyze the temporal and spatial expression patterns of LsRACK1 in SBPH,and the result demonstrated that the expression level of LsRACK1 was highest in four-instar nymph stages and testis.LsRACK1 expression in viruliferous and non-viruliferous adult planthoppers was analyzed though qRT-PCR and Western blot,and the results indicated that there were no different in transcriptional and protein level in response to RBSDV infection.Co-localization of RBSDV CP and LsRACK1 in midgut,ovarian follicular cells and testis of SBPH was observed by tissue immunofluorescence.Laser confocal microscopy observation revealed that RBSDV CP co-localized with endoplasmic reticulum marker HDEL-mCherry and formed granular aggregates in Sf9 insect cells,whereas LsRACK1 did not co-localized with the endoplasmic reticulum marker and mainly localized in cytoplasm and cell membrane,and part of them formed clusters in Sf9 cells.However,when RBSDV CP and LsRACK1 were co-expressed in Sf9 cells,they were found to be co-localized in the granular structure of cytoplasm.The membrane suspension experiment indicated that the interaction between RBSDV CP and LsRACK1 occured on the endoplasmic reticulum,and RBSDV CP can alter the localization of LsRACK1 through transferring LsRACK1 from cytoplasm and cell membrane to endoplasmic reticulum.It is known that RACK1 is the receptor of protein kinase C(PKC)and regulates the translocation and activity of PKC.PKC activity in the viruliferous and non-viruliferous planthopper was detected and found that PKC activity in viruliferous SBPH was remarkable lower than that in non-viruliferous SBPH.Protein kinase C activity in Sf9 cells expressing RBSDV CP or muCP-N were lower than the control expression of GFP,indicating that expression of RBSDV CP and muCP-N attenuated PKC activity.In order to further investigate the effect of PKC kinase activity on rival infection,the PKC specific inhibitors or the activator were injected into the planthoppers to inhibit or activate PKC activity,respectively.After virus feeding,RBSDV in planthoppers was detected.The results demonstrated that inhibitor treatment causes an increase of virus accumulation in planthoppers,while less accumulation of virus was observed in activator treatment planthoppers.Besides,silencing of LsRACK1 induced by dsRNA resulted in decline of PKC activity,and caused more higher viruliferous percentage of SBPH.These results indicated that LsRACKl was involved in the antiviral response of insect vectors.RACK1 enhances PKC mediated phosphorylation in normal cells and we speculate that the decrease of PKC activity in the viruliferous insect may be due to the interaction between CP and LsRACK1,which alters the initial localization and causes function suppression of LsRACK1,finally results in a weakened activity PKC.N-terminal 1-270 amino acid of RBSDV CP was the key site affecting LsRACK1 function and inhibiting PKC activity.3)Expression of RBSDV CP induces autophagyAutophagy is a highly conserved recycling process of intracellular protein,it may be involved in viral infection.In this study,the time-course of autophagy activation in the midgut of L.striatellus in response to RBSDV infection was observed by laser confocal and electron microscopy.The results demonstrated that significantly presence of autophagy in the midgut epithelial cells at 2 days post virus feeding.The levels of autophagy reduced and the virus accumulation simultaneously increased in SBHP at 4 days post virus feeding.Autophagy in SBPH was activated by membrane feeding on activator rapamycin and we found the virus accumulation level was significantly lower in rapamycin treatment than that of the control which was feeded with artificial medium.In addition,in autophagy related gene(ATG3,ATG5 and ATG8)silenced and autophagy inhibitor 3-MA feeding SBPHs,the accumulation level of virus was remarkable increased compared with the control group.These results show that autophagy is involved in the antiviral effect of L.striatellus and autophagy may play an important role in insect resistance to virus infection.To determine whether RBSDV CP is a viral factor inducing autophagy in SBPH,RBSDV CP was expressed in Sf9 cell and transmission electron microscope observation shown that presence of autophagy in CP-expressed Sf9 cell.Several autophagy related genes were amplified from SBPH and yeast two-hybrid assay revealed that RBSDV CP could interacts with ATG3.These results suggest that RBSDV CP is a viral inducer of autophagy in SBPH.In summary,the interaction between RBSDV CP and L.striatellus is a complex biological process.On one hand,RBSDV CP interacts with LsRACKl and alters the location of LsRACKl,which attenuated PKC kinase activity to inhibit the defense response of insect vector.On the other hand,the expression of RBSDV CP induces autophagy in insect cells and then the immune response of insect cells is activated in response to viral infection.