The Mechanism Of Interaction Between Forest Strawberry Chlorophyll A/b Binding Protein LHC Ⅱ And Movement Protein P1 Encoded By Strawberry Vein Banding Virus

Strawberry vein banding virus（SVBV）is a member of the genus Caulimovirus infecting Fragaria species.The results show that ORF I is a movement protein related with intracellular and intercellular movementt and long-distance transport which encoded by Strawberry vein banding virus.But how host factor LHC Ⅱ interacted with movement protein P1,which affects virus transport is unclear.A yeast two-hybrid c DNA library of high viruliferous strawberry was constructed.To find the special receptors in interaction with SVBV,SVBV P1 was used as a bait to screen the library in this paper.This topic on the basis of the study,host factor LHC Ⅱ interacted with movement protein P1.The interaction between the full-length LHC Ⅱ and P1 protein was confirmed by Y2H,BiFC,pull-down and Co-IP.We identificated that LHC Ⅱ protein promotes P1 protein moving and use SVBV P1 protein can be complementary movement deficient PVX vector.This work can provide theoretical basis for further exploring the molecular interaction mechanism between SVBV and woodland strawberry.1.Screening of the host factors of woodland strawberry interacting with P1 of Strawberry vein banding virus by yeast two-hybrid systemY2H competent cells which contained pGBKT7-P1 plasmids were prepared in large scale,and then the cDNA library vectors were transformed into Y2H competent cells.The transformants were incubated on SD/-Trp/-Leu mediumand SD/-Trp/-Leu/-His/-Ade/-Gluc medium.yeast colonies which grew well and changed blue on medium were cultured in liquid medium,and plasmids in these colonies were extracted.The plasmids were transformed into E.coli DH5a cells.The colonies were assigned as positive colonies,plasmids in these colonies were extracted and sequenced.Sequences of strawberry in positive vectors were blasted in Genbank database.The results,these cDNA strawberry belonged to 15 different gene sequences,and three host factors with high abundance of screening.Meanwhile LHC Ⅱ gene screening ratio of 10%,The results Indicated that LHC Ⅱ and P1 are more closely related and important.2.Analysis relative expression levels of LHC Ⅱ in strawberry of SVBV infectious clones by real-time RT-PCRLHC Ⅱ expression was observed in strawberry of SVBV infectious clones when evaluated by real-time RT-PCR.Real-time RT-PCR results showed that LHC Ⅱ gene accumulation levels increased significantly after the SVBV infection,more than 1.5times for strawberry in health.3.The subellular localization and co-localization of SVBV P1 and LHC Ⅱ proteinthe transient expression of P1-GFP and LHC Ⅱ-mCherry fusion protein in N.benthamiana cells was achieved through agro-infiltration.Green fluorescence from P1-GFP fusion protein observed in cytoplasm,mCherry fluorescence from LHC Ⅱ-mCherry fusion protein observed in cytoplasm and Nuclei.Meanwhile co-localization of SVBV P1 and LHC Ⅱ protein in cytoplasm.4.The interaction between the LHC Ⅱ and P1 protein was confirmed by yeast two-hybrid systemY2H competent cells which contained pGBK-P1 and pGAD-LHC Ⅱ,pGBK-LHC Ⅱ and pGAD-P1 plasmids were prepared inY2H competent cells.The transformantswereincubatedonSD/-Trp/-Leumediumand SD/-Trp/-Leu/-His/-Ade/-Gluc medium.The results found that only BK-P1 and AD-LHC Ⅱ transformed yeast can grow blue yeast fell.It suggested that the interaction between the LHC Ⅱ and P1 protein,but others can not.5.The interaction between the LHC Ⅱ and P1 protein was confirmed by bimolecular fluorescence complementation systemYFP^n-P1 and YFP^c-LHC Ⅱ,YFP^n-LHC Ⅱ and YFP^c-P1 in N.benthamiana cells were achieved through agro-infiltration.The fluorescence showed that these two protein interacted in cytoplasm and stomata,but others can not.6.GST pull-down assays the interaction between LHC Ⅱ and P1 proteinTaking the antibody incubation flag into MBP-P1 protein and LHC Ⅱ-flag protein,eluting the excess protein bound on the beads,eluting protein was detected by western blot.The result shows MBP-P1 protein was detected in eluting protein,but others can only detecte LHC Ⅱ-flag protein and no MBP-P1 protein,GST pull-down verify the interaction between LHC Ⅱ and P1 protein in vitro.7.CO-IP assays the interaction between LHC Ⅱ and P1 proteinthe transient expression of p1307-6*myc and p1307-LHC Ⅱ-3*flag fusion protein in N.benthamiana cells was achieved through agro-infiltration.Taking the antibody incubation flag into myc-P1 protein and LHC Ⅱ-flag protein,eluting the excess protein bound on the beads,eluting protein was detected by western blot.The result shows myc-P1 protein was detected in eluting protein,but others can only detecte LHC Ⅱ-flag protein and no myc-P1 protein.CO-IP assays in vivo the interaction between LHC Ⅱ and P1 protein.8.LHC Ⅱ protein promotes P1 protein moving and uses SVBV P1 protein can be complementary movement deficient PVX vectorWe identificated that LHC Ⅱ protein promotes P1 protein moving and use SVBV P1 protein can be complementary movement deficient PVX vector.A.tumefaciens cells containing the plasmids PVX（?P25）-GFP(OD₆₀₀=0.001),PVX（?P25）-GFP(OD₆₀₀=0.001)and pCAM(OD₆₀₀=1.0),MMA,PVX（?P25）-GFP(OD₆₀₀=0.001)and P25(OD₆₀₀=1.0),PVX（?P25）-GFP(OD₆₀₀=0.001)and pCAM–myc-P1(OD₆₀₀=1.0),PVX（?P25）-GFP(OD₆₀₀=0.001)and pCAM–myc-P1(OD₆₀₀=1.0)and pCAM–flag-LHC Ⅱ(OD₆₀₀=1.0)were infiltrated into N.benthamiana cells.The results,these show that P1 and P25 can be complementary movement deficient PVX vector,and the green fluorescences exist from single cell to two-four cells.But LHC Ⅱ and P1and PVX（?P25）-GFP which the green fluorescence mang cells.The results show that LHC Ⅱ is a function protein which involved in viral movement.