Function Domain Analysis And Intracellular Movement Mechanism Of Movement Protein Of Strawberry Vein Banding Virus

Plant virus enter into host and bulk copies,in order to establish a successful systemic infection,plant virus have to overcome intracellular and intercellular lateral movement and long-distance radial transport by vascular bundle.It involves in virus self-encoded specific movement protein.The results show that ORF I is a movement protein which encoded by Strawberry vein banding virus.But how SVBV intracellular and intercellular movementt and long-distance transport in host is unclear.This subject has studied the movement mechanism of MP protein of Strawberry vein banding virus.we analyzed and identified MP protein function domains.And we have studied the intracellular movement mechanism of SVBV MP protein.It provides the theory for the further research of SVBV MP protein.1.Prediction of SVBV MP protein function domainsUsing the software of predicting protein function domain.we have found that the amino acids residues 119-233 is involved in viral movement.,And the amino acids residues 30-55 and 283-329 is the key domain of protein interaction.we designed three deleted mutants MPΔ30-55,MPΔ119-233 and MPΔ283-329.2.Identification the the movement domain of SVBV MP mutant proteinTo determined the function domains of MP.we designed three deletion mutants MPΔ30-55,MPΔ119-233 and MPΔ283-329.（1）Subcellular localization of deletion mutant MPA.tumefaciens cells containing the plasmids pCV-MP-YFP,pCV-MP^?30-55-YFP,pCV-MP^?119-233-YFP,pCV-MP^?283-329-YFP and pCV-YFP were infiltrated into N.Benthamiana.The results show that MP,MP^?30-55 and MP^?283-329 located in cell wall.MP^?119-233 exist in the cytoplasm and nuclei.All the results suggest that the amino acids residues 119-233 is a function domain which involved in viral movement.（2）Covering function of deletion mutant MPA.tumefaciens cells containing the plasmids p PVX-?P25-GFP,p PVX-?P25-GFP+p Bin438,p PVX-?P25-GFP+p Bin-P25,p PVX-?P25-GFP+p Bin-MP^?30-55,p PVX-?P25-GFP+p Bin-MP^?119-233 and p PVX-?P25-GFP+p Bin-MP^?283-329 were infiltrated into N.Benthamiana.The results show that MP^?30-55,MP^?283-329 and P25 can be complementary movement deficient PVX vector,and the green fluorescence exist from single cell to three-four cells.But MP^?119-233 and p Bin438 which the green fluorescence only in single cell.The results show that the amino acids residues 119-233 is a function domain which involved in viral movement.3.Verification the function domain of SVBV MP and P4 protein interaction（1）Verification the domain about protein interaction by the yeast two-hybrid Plasmids p GBK-MP^?30-55+p GAD-P4 and p GBK-P4+p GAD-MP^?30-55 and p GBK-MP^?119-233+p GAD-P4 and p GBK-P4+p GAD-MP^?119-233 and p GBK-MP^?283-329+p GAD-P4 and p GBK-P4+p GAD-MP^?283-329 were cotransformed into yeast.The experiment results show that plasmids p GBK-P4+p GAD-MP^?119-233 were cotransformed into yeast cells which has a capability of growing on selective media.Indicating that amino acids residues 30-55 and 283-329 is a function domain which involved in protein interaction.（2）Verification the domain about protein interaction by bimolecular fluorescence complementation A.tumefaciens cells containing the plasmids pCV-nYFP-MP^?30-55+pCV-c YFP-P4 and pCV-nYFP-P4+pCV-c YFP-MP^?30-55 and pCV-nYFP-MP^?119-233+pCV-c YFP-P4 and pCV-nYFP-P4+pCV-c YFP-MP^?119-233 and pCV-nYFP-MP^?283-329+pCV-c YFP-P4 and pCV-nYFP-P4+pCV-c YFP-MP^?283-329 were infiltrated into N.Benthamiana.The results show that only fluorescence can be seen in N.benthamiana leaves epidermal cells with A.tumefaciens cells containing plasminds pCV-nYFP-MP^?119-233+pCV-c YFP-P4.All the results suggest that amino acids residues 30-55 and 283-329 is a function domain which involved in protein interaction.4.Intracellular subcellular localizations of SVBV MPA.tumefaciens cells containing the plasmids pCV-MP-YFP was infiltrated into N.benthamiana.The results show that the punctate spots form by SVBV MP exist in the cytoplasm and nuclei in N.benthamiana epidermal cells.What’s more,the punctate spots MP can along a certain trajectory to the cell wall,and finally located in the plasmodesmata（PD）.The results show that the punctate spots form by SVBV MP along a certain trajectory target to PD.5.SVBV MP co-localization with microtubulesA.tumefaciens cells containing the plasmids pCV-MP-YFP and pCV-MT-RFP were infiltrated into N.benthamiana.The results show that SVBV MP co-localization with microtubules（MT）.After treatment by microtubule inhibitor,then infiltrated A.tumefaciens cells containing the constructs pCV-MP-YFP and pCV-MP-YFP+pCV-MT-RFP.It shows that SVBV MP co-localization with microtubules（MT）,only the punctate spots target to Plasmodesmata is reduced.All the results indicate that SVBV MP target to plasmodesmata related to cytoskeletonmicrotubules.6.SVBV MP co-localization with actinA.tumefaciens cells containing the plasmids pCV-MP-YFP and p CAM-ABD2-GFP were infiltrated into N.benthamiana.The results show that SVBV MP co-localization with actin（ABD2）,indicating that SVBV MP target to plasmodesmata related to actin.7.SVBV MP co-localization with endoplasmic reticulumA.tumefaciens cells containing the plasmids pCV-MP-YFP and pCV-ER-RFP were infiltrated into N.benthamiana.The results show that SVBV MP co-localization with endoplasmic reticulum（ER）,indicating that SVBV MP target to plasmodesmata related to endoplasmic reticulum.8.Interaction of protein which encoded by SVBVPlasmids p GBK-P3+p GAD-P4 and p GBK-P4+p GAD-P3 were cotransformed into yeast.The results suggest that SVBV P3 and P4 protein interaction.A.tumefaciens cells containing the plasmids pCV-nYFP-P2+pCV-c YFP-P3 and pCV-nYFP-P3+pCV-c YFP-P2 and pCV-nYFP-P3+pCV-c YFP-P4 and pCV-nYFPP4+pCV-c YFP-P3 were infiltrated into N.benthamiana.The results suggest that SVBV P P2/P3 and P3/P4 protein interaction.All the results show that SVBV nucleocapsid protein（P4）and P3 protein interaction,aphid transmission protein（P2）and P3 protein interaction,then the virus enter into host.Virions movement in the cell by P4 / MP.