Study On The Interaction Of Cucumber Mosaic Virus Coat Protein With Tomato Photosynthesis Related Proteins

Cucumber mosaic virus(CMV)belongs to Bromoviridae and the typical species of Cucumovirus.The coat protein(CP)is located at the outmost layer,which is an important symptom determining factor associated with long-distance movement and aphid transmission.Photosynthesis is one of the most important physiological and biochemical activities of green plant,which is mainly regulated and controlled by chloroplast proteins.The invasion of plant virus results in typical symptoms of plant leaves and disordered functions of chloroplast proteins.Deeply understanding and researching the interactions between viral proteins and chloroplast proteins is of important significance for revealing the pathogenesis of plant virus diseases as well as antiviral mechanism.Previous research of our laboratory used CMV CP as a bait to screen out 9 host proteins related to photosynthesis,and 6 genes were selected for further verification.The results are as follows:The total RNA of tomato healthy leaves was used as the template to clone complete code sequences(CDS)of the 6 proteins by RT-PCR which were successfully cloned into the prey vector pPR3-N and respectively transformed into NMY51 with CMV CP bait vector,then samples were diluted and plated on three types of selective medium.The results showed that Ribulose-1,5-bisphosphate carboxylase small chain 2a(clone number E104),Ribulose-1,5-bisphosphate carboxylase small chain 3b(clone number B3),10KDa peptide of photosystem ? PsbR(clone number E57),and Photosystem ?core complex protein PsbY(clone number E161)are interacted with CMV CP while Photosystem ? core complex protein PsbW(clone number D15)and Protochlorophyllide reductase are not.According to bimolecular fluorescence complementation assay(BIFC),vectors Y~N-CMV CP and Yc-B3,Yc-E57,Y~C-E104,Yc-E161 were constructed.All the vectors were transformed into agrobacterium and Y~C-B3,Yc-E57,Y~C-E104,Yc-E161 were respectively injected into Nicotiana benthamiana with Y~N-CMV CP and all treatments have yellow fluorescence observed by the laser scanning confocal microscope.The results also show that CMV CP interacts with the four proteins refered before.In order to determine the interaction domains of CMV CP preliminary which were interated with the four proteins,the gene of CMV CP was divided into three segments and they are 1-327bp(CMV CP-1),163-489bp(CMV CP-2)and 328-654bp(CMV CP-3).Then the three segments were cloned into the vetor of BIFC separately named as Y~N-CMV CP-1,Y~N-CMV CP-2,Y~N-CMV CP-3,and all the vectors were transformed into agrobacterium and separately injected into Nicotiana benthamiana with Y~C-B3,Y~C-E57,Y~C-E104 and Y~C--E161.Laser scanning confocal microscope was used to observe the fluorescence of the hypodermis.The results showed that Y~N-CMV CP-1,2,3 and Yc-B3;Y~N-CMV CP-1,3 and Y~C-E57;Y~N-CMV CP-1,2,3 and Y~C-E104;Y~N-CMV CP-1,2 and Y~C-E161 had yellow fluorescence;Y~N-CMV CP-2 and Y~C-E57 Y~N-CMV CP-3 and Y~C-E161 didn't have yellow fluorescence.So the results illustrate that CMV CP has multiple interaction domains which can interact with different host proteins.Then analyse the probable amino acid sites of interaction domains of CMV CP and used homologous modeling softwares to simulate the secondary structure and tertiary structure of CMV CP,CMV CP-1,CMV CP-2,CMV CP-3.By analyzing the secondary structure,the tertiary structure and the physicochemical property of CMV CP-1,CMV CP-2,CMV CP-3,it was inferred that the 76aa-84aa?127aa-136aa?154aa-164aa?189aa-199aa of CMV CP may be the active interaction sites and they are all ? turns.This study proved that the four host proteins related to photosynthesis interacted with CMV CP and predicted the probable interaction sites.It could lay a solid foundation for further studies of CMV pathogenic mechanisms.