Identification And Characterization Of Proteins Interacting With Rice Grass Stunt Virus In Host Plants

Rice grass stunt virus(RGSV)is a member of the genus Tenuivirus,which includes Rice stripe virus(RSV)as the type species,and is transmitted by a brown planthopper in a circulative and propagative manner.The RGSV RNAs,all of which are ambisense,are called RNA1 to RNA6 in the decreasing order of their size.RGSV has caused a destructive disease of rice in Southeast countries including Indonesia,Philippines,Vietnam,and several provinces of China such as Fujian,Guangdong,Guangxi,Hainan and Taiwan.Typical symptoms induced by RGSV infection are leaf yellowing(chlorosis),stunting,and excess tillering(branching),usually affected rice plants are unable to flower.All these suggest that RGSV infection affects rice growth and development substantially.In spite of intensive studies in recent decades on the pathogenesis,molecular biology,genetic diversity,and control of RGSV,there are still major gaps in our knowledge about the virus,such as the function of RGSV proteins,pathogenic mechanism,molecular mechanisms of plant resistance to RGSV,and,especially the molecular mechanisms of virus symptom development,such as K+-deficient symptom.Therefore,the study on the interaction relationships among viral proteins,and host proteins with viral proteins at molecular level,has an important significance.Protein-protein interactions(PPIs)play a crucial role in many cellular processes.Identification and characterization of PPIs can provide important insights into protein functions at the cell level.For viruses,mapping the interaction networks of viral proteins contributes considerably to understanding the mechanisms of viral pathogenicity,the virus infection cycle,and virus-host interactions.The main results were summarized as follows:1.Mapping the interaction networks of RGSV proteinsTen genes of RGSV were amplified by RT-PCR and inserted into Y2H bait vector and prey vector respectively.In order to detect the interaction between the proteins,the recombinants were transformed into yeast strain Y2HGold two by two,and the transformants were plated on the different synthetic dropout nutrient mediums.The results showed:p1,p2,p3,pC5 can interact with themselves,and interactionsbetween different proteins were:p1 with p2,p3,p4 and p5;p2 with p3 and p5;p3 with p4 and pC4;p4 can interact with P5;These interactions were confirmed by a bimolecular fluorescence complementation(BiFC)assay in Nicotiana benthamiana cells.2.Host proteins interacting with RGSV P2 and P5 were identified and characterized.In order to study the interaction between the virus and host plant,p3 and p5 of RGSV were used as bait proteins to screen cDNA Library of nicotiana benthamiana by Y2H.Sixty and seven proteins were obtained for p3 and p5,respectively.Consistent with a role of P3/P5 in repressing host defense responses including RNA silencing,most of the host proteins interacting with P3/P5 are associated with the GO biological process terms response to abiotic and biotic stimulus.Furthermore,many of the host proteins interacting with p3 were chloroplast genes,these interactions may be responsible for the yellowing symptom associated with RGSV infection.Meanwhile,there are two host proteins,NbArfGAP and OsSty-2,interacting with p5 regulating endocytosis and the ability of plant virus movement proteins to alter plasmodesmata to promote virus cell-to-cell transport,predicting that p5 may involve in viral cell-to-cell movement.3.The interactions between OsCBLl/OsCIPK5/OsCIPK25 and mulitiple proteins encoded by RGSV/RSVThe results showed:OsCIPK5 and OsCIPK25 could interact with pi,p2 and p5 by Y2H and BiFC assay.The RNA silencing suppressor proteins,NS2 and NS3,of RSV could also interact with OsCIPK5 and OsCIPK25 by BiFC assay in Nicotiana benthamiana cells.Further studies showed that p3 not only can interact with OsCBLl,but also interact with OsCIPK23 and OsAKT1.All of the three proteins,OsCBLl,OsCIPK23 and OsAKT1,are important protein components of the conserved K+uptake pahway.It was indicated that p3 interacting with those proteins affected K+uptake in plants and was involved in K+-deficient symptom development.4.OsCBLl/OsCIPK5/OsCIPK25 involved in K+-deficient symptom development(1)Analysis of functional domain of OsCIPK5 and OsCIPK25:The bioinformatics analysis showed that OsCIPK5 and OsCIPK25 contained two functional domains,N-terminal kinase activation loop domain and C-terminal NAF domain,which similar to other CIPKs that had been reported.(2)Enzymatic activity assay:In vitro phosphorylation assays were performed to detect the enzymatic activity of target kinase proteins.The results showed that auto-phosphorylation of OsCIPK5 and OsCIPK25 cannot be activated by Mg2+while Mn2+and Ca2+only had a weak effect on OsCIPK5.Interestingly,strong activity was observed in the presence of Mn2+and Ca2+for OsCIPK25.(3)analysis of Expression patterns of kinase genes under biotic and abiotic stress treatments:Real-time quantitive PCR was used to analyze the expression patterns of kinase genes.The expression of OsCBL1,OsCIPK23,OsAKT1,OsCIPK25 and OsCIPK5 were up-regulated by RGSV infection;Meanwhile OsCIPK25 was induced by lower K+treatment in roots and leaves.However,the expression of OsCIPK5 was only induced under lower K+in roots.(4)The K+content in RGSV infected rice was reduced significantly.(5)Morever,OsCIPK5 RNAi rice plants induced unfilled grain,while the phenotype of OsCIPK25 RNAi rice plants in T1 generation are leaf yellowing(chlorosis)and stunting.All of these evidence indicated that OsCIPK5,OsCIPK25 were involved in rice K+uptake and K+-deficient symptom development...