Study On The Interaction Among N,Nsm And Nss Proteins Encoded By Tomato Zonate Spot Virus

Tomato zonate spot virus(TZSV)is a dominant species of Orthotospovirus of the Tospoviridae family,and of the Bunyavirales order found in Yunnan Province.The virus infects and harms important cash crops such as tomatoes Nicotiana tabacum,Lycopersicon esculentum,Capsicum annuum and flowers,and have spread to the surrounding areas of Yunnan in recent years.The TZSV genome consists of three single-stranded RNAs,namely RNA L,RNA M,and RNA S.At present,many scholars have studied TZSV from the aspects of biology and molecular biology,and clarified the pathogenic nature,geographical distribution,symptoms,pathogenic differentiation,serology,cytopathology,etc.of TZSV.However,the function of some proteins encoded by the virus,the pathogenic mechanism of the virus,and the mechanism of the host's resistance to the virus are still unclear.Therefore,studying the interaction between viral proteins at the molecular level is of great significance for elucidating many of the above-mentioned problems.In order to analyze the interaction between the proteins encoded by TZSV,this paper studied the interaction between the three proteins N,NSm and NSs encoded by TZSV through yeast two-hybrid in and bimolecular fluorescence complementation(BiFC)technology.The three proteins of N,NSm and NSs were analyzed for subcellular localization and colocalization in plant cells.First,the yeast recombinant plasmid was detected for toxicity to yeast Y2 HGold,and then the yeast two-hybrid system(Y2H)was used to verify the self-interaction and pairwise interaction of N,NSm and NSs encoded by TZSV.The results showed that the three proteins of TZSV N,NSm and NSs showed no toxicity to yeast Y2 HGold.Y2H results show that both N and NSm proteins interacted with themselves,In addition,NSs protein itself has no detected interaction,and no interaction was detected between N and NSm,N and NSs,and NSm and NSs proteins.Since the yeast two-hybrid system has certain false positives,the BiFC further verified the interaction between the three proteins of N,NSm and NSs.The recombinant expression vectors n YFP-NSm,c YFP-NSm,n YFP-N,c YFP-N,n YFP-NSs,and c YFP-NSs were constructed.The results of BiFC showed that: TZSV N and NSm proteins can interact with each other,and N protein self-interaction exists on the cytoplasm and nucleus,and the NSm protein self-interaction forms fluorescent aggregates in the cytoplasm.Interactions between N and NSm,N and NSs,or NSm and NSs can also be detected.The interaction between N and NSm exists on the cytoplasm and nucleus,and the interaction between N and NSs is located on the cytoplasm and nucleus.The interaction between NSm and NSs protein is located on the cytoplasm,while the control group has no yellow fluorescence.To further analyze the localization and co-localization of N,NSm and NSs proteins encoded by TZSV,and the subcellular localization expression vectors NSm-m NG,N-m NG,NSs-m NG,NSm-m Cherry,N-m Cherry and NSs-m Cherry were constructed using the method of homologous recombination.The subcellular localization results showed that: N protein is localized in the cytoplasm and nucleus;Both NSm and NSs proteins are located in the cytoplasm.Co-localization analysis of the interactions between N and NSm,N and NSs,and NSm and NSs proteins,N-m NG and NSs-m Cherry are co-localized on the cytoplasm;N-m Cherry and NSm-m NG are co-localized on the cytoplasm;NSm-m NG and NSs-m Cherry are co-localized on the cytoplasm.And the results were consistent with the results of BiFC.Therefore,it can be concluded that N and NSm proteins have self-interaction phenomenon,and there is pair interaction phenomenon among N,NSm and NSs proteins.These results lay the foundation for further research on the interaction between viral proteins and the molecular mechanism of viruses.