Effect Of Strawberry DnaJ Protein On The Ability Of Strawberry Vein Banding Virus P1 Protein To Promote Virus Infection

Strawberry vein banding virus（SVBV）is the most important disease in strawberry planting industry,which seriously endangers the yield and quality of strawberry.Previous studies have proved that SVBV P1 protein is a movement protein,which is closely related to the transfer and diffusion of the virus.However,how P1 protein interacts with host factors to affect intercellular movement and systemic infection of virus has not been reported.Our research group constructed a c DNA library of forest strawberry in the early stage,and screened the heat shock protein DnaJ interacting with P1 protein as bait.DnaJ protein is an important molecular chaperone and participates in many physiological processes of plant cells.It may be an important host factor regulating the function of P1 protein.Firstly,yeast two hybrid,bimolecular fluorescence complementary（BiFC）,Co-IP and firefly luciferase fragment complementary imaging（LCI）experiments were used to prove that P1 can interact with DnaJ in vitro and in vivo.Subcellular localization found that P1 was located in the cytoplasm and cell edge,DnaJ was located in the chloroplast,and both were located at the cell edge,indicating that DnaJ may directly or indirectly target P1.DnaJ did not affect the localization of P1 in endoplasmic reticulum and plasmodesmata.SVBV infection of forest strawberry can induce the down-regulation of DnaJ expression,indicating that DnaJ is involved in the process of virus infection.DnaJ protein can inhibit the ability of P1 protein to replenish the intercellular movement of motion deficient PVX,and DnaJ can also inhibit the ability of P1 protein to replenish the movement of motion deficient CMV system.It was found that overexpression of DnaJ could inhibit the infection rate of SVBV.1 Interaction between SVBV P1 and FvDnaJThe interaction between P1 and FvDnaJ was verified by yeast two hybrid AD-P1and BK-DnaJ co-transformed yeast competent cells for yeast two hybrid test.The results showed that P1 and FvDnaJ co-transformed yeast could grow in SD/-Trp/-Leu/-ADE/-His solid medium,which was similar to the positive control,indicating that P1 could interact with FvDnaJ in vitro.The interaction between P1 and FvDnaJ was verified by BiFC nYFP-P1 and cYFP-DnaJ,nYFP-DnaJ and cYFP-P1 were co-infiltrated into N.benthamiana,respectively.The green fluorescence was observed in the leaves of N.benthamiana co-infiltrated with nYFP-P1 and cYFP-DnaJ or nYFP-DnaJ and cYFP-P1 by laser confocal microscope,indicating that P1 can interact with FvDnaJ in vivo.The interaction between P1 and FvDnaJ was verified by Co-IP Myc-DnaJ and P1-YFP were co-infiltrated N.benthamiana.The total protein of infiltrating leaves was extracted for immunoprecipitation test.The results showed that DnaJ protein could bind P1 protein,indicating that P1 could interact with FvDnaJ in vivo.The interaction between P1 and FvDnaJ was verified by LCI P1-nLuc and DnaJ-cLuc were co-infiltrated N.benthamiana,and the infiltrated leaves were sprayed with chromogenic substrate.The fluorescence was observed in the leaves of P1-nLuc and DnaJ-cLuc co-infiltrated N.benthamiana by plant in vivo imager,which was similar to the positive,indicating that P1 could interact with FvDnaJ in vivo.2 Subcellular localization and co-localization of SVBV P1 protein and FvDnaJP1 or FvDnaJ was infiltrated N.benthamiana respectively.The results showed that P1 waslocated in the cytoplasm and cell periphery,and FvDnaJ was located in the chloroplast.P1and FvDnaJ were co-infiltrated N.benthamiana leaves.The results showed that P1 and FvDnaJ were co-localized at the edge of N.benthamiana epidermal cells.3 FvDnaJ Gene Expression is downregulated in SVBV-infected F.vescahealthy F.vesca was inoculated with A.tumefaciens containing the infectious clone of SVBV using the vacuum-infiltration method,Compared with the mock control,F.vesca inoculated with the infectious clone of SVBV showed vein banding symptoms at 35 dpi,the detection showed the transcription level of FvDnaJ was significantly downregulated by q PCR assay.4 Effect of FvDnaJ on P1 protein functionFvDnaJ did not affect the localization of P1 in plasmodesmata and endoplasmic reticulum P1 and FvDnaJ were co-infiltrated N.benthamiana with plasmodesmata or endoplasmic reticulum marker protein respectively.The results showed that P1 could co-locate with PDLP5 or ER marker protein,indicating that FvDnaJ did not affect the localization of P1 in plasmodesmata and endoplasmic reticulum.FvDnaJ inhibits P1 to complement movement deficient PVX movement deficient PVX^ΔP25-GFP,P1 and DnaJ were co-infiltrated N.benthamiana.It was observed that the number of green fluorescent cells in infiltrated N.benthamiana leaves decreased,indicating that FvDnaJ can inhibit P1 to complement the intercellular movement of movement deficient PVX.Acquisition of FvDnaJ transgenic N.benthamiana The plant expression vector p CM-1307-6*Myc-FvDnaJ was constructed.FvDnaJ was inserted into the genome of wild type N.benthamiana by leaf disc method to obtain F₀ generation FvDnaJ transgenic N.benthamiana.The positive plants were screened by RT-PCR,and finally the stable F₃generation FvDnaJ transgenic N.benthamiana was obtained.The results of RT-PCR and Western blot were correct.SVBV P1 complements the systemic movement of movement defective CMV Construct vector CMV^ΔMP-P1 was inoculated with N.benthamiana.After 8 days,it was observed that the systemic leaves of N.benthamiana had slight mosaic symptoms.The accumulation of CMV CP was detected in CMV^ΔMP-P1-infected N.benthamiana systemic leaves by western blot,indicating that P1 was able to complement the systemic movement of movement defective CMV.FvDnaJ inhibits P1 to complement movement deficient CMV CMV^ΔMP-P1 was inoculated with wild-type N.benthamiana and FvDnaJ transgenic N.benthamiana,respectively.After 8 days,it was observed that mild mosaic symptoms in infected FvDnaJ transgenic N.benthamiana.The accumulation of CMV CP protein in the systemic leaves of infected FvDnaJ transgenic N.benthamiana was decreased by western blot assay.CMV^ΔMP-P1 was inoculated with wild-type N.benthamiana and FvDnaJ transgenic N.benthamiana respectively.After 3 days,the expression of CMV CP protein in inoculated leaves was detected by western blot assay.The results showed that the accumulation of CMV CP protein in wild-type N.benthamiana and FvDnaJ transgenic N.benthamiana was the same,indicating that FvDnaJ did not affect CMV^ΔMP-P1 replication,FvDnaJ can inhibit P1 to complement the systematic movement of mobement deficient CMV.5 Overexpression of FvDnaJ inhibits F.vesca infection by SVBVOverexpression of FvDnaJ by TRV virus vector The recombinant vector TRV-FvDnaJ-Myc was constructed and infected F.vesca by vacuum filtration method.After 15days,The detection showed that the transcription level of FvDnaJ in TRV-FvDnaJ-Myc-infected F.vesca seedlings was up-regulated by q PCR assay.Overexpression of FvDnaJ can inhibit SVBV infection TRV-FvDnaJ-Myc-infiltrated plants were infiltrated with the infectious clone of SVBV.After 35 days,the detection showed that the accumulation of SVBV CP protein in F.vesca seedlings was decreased by western blot assay,indicating that overexpression of FvDnaJ can inhibit F.vesca infection by SVBV.