Molecular Mechanism Of Laodelphax Striatellus Cell Autophagy Induced By Rice Black-streaked Dwarf Virus

Rice(Oryza sativa)is the most important food crop worldwide.However,rice black-streaked dwarf virus(RBSDV)transmitted by Laodelphax striatellus(small brown planthopper)in a persistent and propagative manner,seriously harms rice plants,and causes significant yield losses of food crops every year.Autophagy is an important innate immunity,and plays important role in resisting virus invasion.Viruses have also evolved mechanisms to avoid and inhibit autophagy for their invasion,and some even utilize the autophagosome membrane and autophagy mechanism of the host to promote their replication and diffusion.However,it is still unknown that whether RBSDV can cause autophagy in insects L.striatellus.What is the role of L.striatellus cell autophagy on RBSDV replication and transimission? What is the molecular mechanism of L.striatellus cell autophagy induced by RBSDV? These scientific questions need urgently to be answered.For this purpose,this present dissertation investigated whether RBSDV infection causes L.striatellus cell autophagy,studied the the molecular mechanism of L.striatellus cell autophagy induced by RBSDV,and analyzed the role of L.striatellus cell autophagy on RBSDV infection and replication.The research results were summarized as follows:(1)RBSDV infection induces L.striatellus cell autophagy and autophaty inhibts RBSDV invasion and replicationAutophagy in RBSDV-infected L.striatellus midgut cells was analyzed using the following three approaches: Western blot analysis of ATG8-II accumulation,electrton microscopy observation of autophagosomes,and immunofluorescence localization of ATG8.We found that RBSDV infection can increase the accumulation of ATG8-II and the number of double-membrane autophagosomes in L.striatellus cells at 2–4 days post feeding on RBSDV-infected rice plants.Confocal microscopic analysis also revealed ATG8 puncta in the RBSDV-viruliferous midgut cells.Theses results suggest that RBSDV infection can induce autophagy in RBSDV-viruliferous L.striatellus at an early infection stage.To investigate the effect of autophagy on RBSDV infection and replication in L.striatellus,we treated L.striatellus with an autophagy inhibitor 3-methyladenine(3-MA),or an autophagy activator rapamycin for 24 h,then the L.striatellus obtained RBSDV by feeding RBSDV-infected rice plants.The results discovered that the levels of RBSDV P10 protein and P10 transcript were significantly increased in the 3-MA-treated L.striatellus,but significantly decreased in the rapamycin-treated L.striatellus.The percentage of RBSDV-viruliferous L.striatellus from ATG3-,ATG5-or ATG8-ds RNA injection treatment was notably higher compared with that of the control.These data indicate that Laodelphax striatellus cell autophagy can suppress RBSDV invasion and replication.(2)RBSDV P10 alone can induce autophagy in Sf9 insect cells and L.striatellusBecause RBSDV infection can induce autophagy in L.striatellus,we decided to investigate if a single RBSDV protein can cause autophagy.We individually expressed all 13 open reading frames(ORFs)of RBSDV in Sf9 insect cells using recombinant baculovirus expression vectors,and then analyzed the cells for autophagy activity using Western blot assays and transmission electron microscope(TEM).We found that the average number of autophagosomes and accumulation of ATG8-II in the RBSDV P10-expressing Sf9 cells was much higher than that in the empty baculovirus vector-transfected or in GFP-transfected Sf9 cells.To determine whether recombinant P10 can also induce autophagy in L.striatellus midgut cells,we analyzed the midguts from L.striatellus fed with an artificial diet containing prokaryotic expressed recombinant RBSDV P10 through TEM and Western blot assay.The result showed that the number of autophagosomes in the midgut epithelial cells of L.striatellus fed with the artificial diet containing P10 was much higher than that in cells of L.striatellus fed with an artificial diet containing GST.In addition,Western blot assays showed that more autophagy was induced in the midguts of L.striatellus fed P10 compared with that in the midguts of L.striatellus fed GST.These results indicate that RBSDV P10 alone can induce autophagy in Sf9 insect cells and L.striatellus.(3)RBSDV P10 can interact with GAPDH and change the subcellular localization of GAPDHTo investigate how RBSDV P10 induces autophagy in L.striatellus,we performed a Y2 H assay to screen an L.striatellus c DNA library.In this screen,a glyceraldehyde-3-phosphate dehydrogenase(GAPDH)-encoded protein was found to interact with RBSDV P10.Both pulldown assay and Co-IP assay further proved the RBSDV P10-GAPDH interaction.To determine the subcellular localization patterns of RBSDV P10 and Ls GAPDH in L.striatellus midgut cells,we allowed L.striatellus to feed on RBSDV-infected rice plants.At different days post feeding,the digestive organ of L.striatellus was collected and analyzed for RBSDV P10 and Ls GAPDH locations using fluorescent antibodes.The confocal microscopy observation discovered that P10 and GAPDH co-localized in the L.striatellus midgut epithelial cells at 4 and 7 dpf,and then the co-localized phenomenon gradually diminished.RBSDV P10 protein also co-localized with GAPDH in midgut epithelial cells after feeding L.striatellus with an artificial diet containing the purified recombinant RBSDV P10 protein.(4)Ls GAPDH can interact with Ls ATG3 and silencing of GAPDH expression affects autophagy occurrenceIt is reported that GAPDH can interact with ATG3 in N.benthamiana cells to suppress autophagy.Our Y2 H,MBP pull-down and Co-IP assay results demonstrated that Ls GAPDH did interact with Ls ATG3.However,silencing of GAPDH expression did not induce autophagy in the Sf9 or L.striatellus cells.Furthermore,silencing GAPDH expression in L.striatellus suppressed autophagy induced by RBSDV infection,indicating that GAPDH plays a completely different role in the autophagy process of insect cells and plant cells.(5)RBSDV P10 induces autophagy by promoting AMPK and GAPDH phosphorylation and subsequent translocation of GAPDH to the nucleusA previous study had indicated that under glucose starvation conditions,cytoplasmic GAPDH is phosphorylated at Ser122 by AMPK,leading to its translocation into the nucleus and phosphorylated GAPDH in the nucleus activates Sirt1 through direct interaction to cause autophagy.In this study,laser confocal microscope obversation and phosphorylation analysis assay found that GAPDH could be phosphorylated and subsequently translocated into the nucleus to activate autophagy in Sf9 insect cells grown on a glucose starvation medium,and the Ser95 is the key phosphorylation site of Ls GAPDH.In addition,both RBSDV infection and RBSDV P10 expression can promote phosphorylation of AMPK,resulting in GAPDH phosphorylation and relocation of GAPDH from the cytoplasm to the nucleus in both L.striatellus midgut cells and Sf9 cells.Thus,the phosphorylated GAPDH in the nucleus activates cell autophagy.Feeding recombinant RBSDV P10 protein also can promte GAPDH to enter the nucleus,and induce L.striatellus cell antophagy,and the silencing of AMPK expression in Sf9 cells inhibits the phosphorylation and redistribution of GAPDH from the cytoplasm to the nucleus caused by RBSDV P10.Furthermore,the phosphorylation level of AMPK protein in viruliferous L.striatellus midgut cells was also significantly increased.In summary,our study has demonstrated that RBSDV P10 can promote Ls AMPK phosphorylation,which leads to Ls GAPDH phosphorylation,and that the phosphorylated Ls GAPDH is translocated from the cytoplasm to the nucleus to activate the autophagy pathway in L.striatellus.Furthermore,autophagy can suppress RBSDV infection and replication in L.striatellus midgut cells.