Construction Of Host Factor Screening System For Turnip Mosaic Virus P1 Protein And Study Its Interaction With Arabidopsis VOZ2 Protein

Potyvirus is the largest RNA viral genus thus far,members of which infect many food and cash crops(grass,medicinal materials,fruit trees)and cause serious economic losses worldwide.The genome of most potyviruses is comprise of a positive-sense single-stranded RNA,which encodes one open reading frame that is translated into a large polyprotein.The polyprotein is hydrolyzed by three viral proteases,namely,P1,Hc Pro and Pro,into 10 mature proteins.P1 is located at the beginning of the viral genome.The N-terminal region of P1 is highly variable,which negatively regulates the self-cleavage and thus the replication of the virus.The C-terminal portion of P1 is relatively conserved and contains a serine protease domain responsible for cis-cleavage of P1-Hc Pro,allowing the release of P1 from the polyprotein.The activation of P1 protease activity depends on unknown host factors that regulate the self-release of P1 from Hc Pro,which is the key to the normal RNA silencing function of Hc Pro.However,the host factor has yet been determined.In this study,we used turnip mosaic virus(Tu MV)and the GAL4/UAS transcriptional activation system to construct a screening system to identify the unknown host factor that required for P1,and analyzed the interaction between Tu MV P1 and its interacting VOZ2 protein obtained in a yeast two-hybrid screening.Detailed research results were as follows:(1)Screening system for P1-interacting host factor: a membrane-anchored35S::6K2-P1-Hc N10-GAL4-VP16/UAS-GFP system was constructed,and the expression of the downstream GFP under UAS in Nicotiana benthamiana was observed by regulating the P1self-cleavage characteristic.It was found that this system could be used as an effective system for screening P1 host factors.Transgenic Arabidopsis plants harboring35S::6K2-P1-Hc N10-GAL4-VP16/UAS-GFP were obtained via agrobacterium-mediated transformation.Homozygous progenies were obtained for constructing mutation library and screening host factor for stimulating the P1 protease activity.No GFP fluorescence was observed on the F2 generation.Immunoblot results suggest that the 6K2-P1-Hc N10-GAL4-VP16 may be silencing in the transgenic plants.To further improve the system,RNA silencing-defective mutants including rdr6,nrpd1 a,and drm1/drm2 were used for producing the transgenic plants.Homozygous transgenic plants in the rdr6 background showed weak GFP signal,indicating RDR6 plays important role in silencing 6K2-P1-Hc N10-GAL4-VP16.Together,these results indicate that the GAL4/UAS transcriptional activation system can be used for screening the host factor for P1,although further optimization is needed.(2)Physical and chemical characteristics of P1 and VOZ2: Comparison of the P1 protein sequences of Tu MV and other three potyviruses by Ja Iview suggested that the N-terminal of P1 is highly variable while the C-terminal is relatively conserved,indicating that the N-terminal plays an important role in regulating the self-cleavage of P1 from Hc Pro;Expasy and Psipred were used to analyze the physicochemical properties and predict the secondary structure of VOZ2.Results showed that VOZ2 is a hydrophilic protein with an intrinsic disordered secondary structure,indicating that VOZ2 is not a membrane protein.Further subcellular localization analyses showed that P1 was located in the nuclei and cytoplasm,and VOZ2 was located in the cytoplasm.(3)Verified the interaction between Tu MV P1 and VOZ2: The interaction between Tu MV P1 and VOZ2 was confirmed by bimolecular fluorescence complementation and yeast two-hybrid.Tu MV-GFP was used to infect Arabidopsis voz2 mutant and quantitative analysis of leaf area showed that the virus accumulation was significantly reduced in voz2 than that in WT.These results suggest that VOZ2 is not the key host factor in regulating the P1 protease activity that required for self-cleavage of P1 from Hc Pro.In conclusion,this study established a screening system of host factor for P1,analyzed the interaction between P1 and VOZ2,and its function in viral proliferation.These results have deepened the understanding of the potyviruses P1 protein,and laid a foundation for screening the host factor required for P1 protease activity.