Strawberry RD21 Degradated SVBV P6 And The Infection Of SVBV Influenced ABA Metabolic Pathway In Strawberry

Strawberry vein banding virus（SVBV）harms strawberry production.Previous studies have found that the silent suppressor P6 of SVBV is closely related to the pathogenicity of the virus.Our research team constructed c DNA of Fragaria vesca library and selected P6as the bait protein,developing out the host factor papain-like cysteine protease——drought response protein 21（RESPONSIVE-TO-DESICCATION-21,RD21）.This study first used genetic transformation technology to obtain FvRD21 transgenic N.benthamiana,Transient expression and systemic expression of P6 in FvRD21 transgenic N.benthamiana were inhibited,revealing that FvRD21 can help host plants resist virus infection.the interaction between FvRD21 and P6 in vivo was verified via bimolecular fluorescence complementary（Bi FC）as well as firefly luciferase complementation imaging assays（LCI）.The genome cloning SVBV inoculated with Fragaria vesca,FvRD21 transcription level increased and ABA metabolic pathway responded,indicating that the genome cloning SVBV infection induced FvRD21-mediated defense mechanism and ABA metabolic pathway in Fragaria vesca;while the genome cloning SVBV inoculated Fragaria vesca,ABA treated the inoculated Fragaria vesca,SVBV virus replication level in vivo is reduced,indicating that ABA can assist host strawberry to resist SVBV infection,which lays the foundation for exploring the defense and anti-defense mechanism between host and virus.1 Obtaining the FvRD21 transgenic plantsConstructing the plasmid 2300s-FvRD21 and using the leaf disc technique,we uccessfully obtained 2 T₀ generation positive strains of FvRD21 overexpressing N.benthamiana.FvRD21 overexpressed N.benthamiana.with high plant height and increased tillering,indicating that FvRD21 can regulate plant growth and development.RT-q PCR technology detected that there was no significant difference in FvRD21expression between the two lines of FvRD21 overexpressing N.benthamiana,indicating that the expression levels of FvRD21 were consistent among the positive lines.2 FvRD21 inhibits SVBV P6 expressionFvRD21 inhibited local expression of P6.P6 respectively co-infiltrated wild-type N.benthamiana with Vec,GST,and RD21-3*flag,Western blot analysis revealed that P6+RD21-3*flag co-infiltration significantly reduced the expression of P6 protein in leaves,showing that FvRD21 can inhibit local expression of P6.P6 was injected with infiltrated wild-type N.benthamiana leaf and FvRD21 overexpressed N.benthamiana leaf,Western blot analysis found that P6 protein of FvRD21 overexpressed N.benthamiana leaf significantly decreased,indicating that FvRD21 can inhibit local expression of P6.FvRD21 inhibits P6 system expression.PVX-P6 respectively infiltrats wild-type B.nicotianae（WT）and FvRD21 overexpressed N.benthamiana（OE-FvRD21）,with the development of the disease course,the expression of P6 protein in WT and OE-FvRD21system leaves both are improved.Under the same number of days,the expression of P6protein in leaves of OE-FvRD21 system was lower than that in leaves of WT system,showing that FvRD21 can inhibit the expression of P6 system and delay the pathogenic course of P6 heterologous aggravation of PVX infection of N.benthamiana.3 Subcellular colocalization of FvRD21 and P6Laser confocal microscopy was used to observe the subcellular localization of P6 and RD21,it was found that P6 was located at nucleus and cytoplasm,and RD21 was located at cell boundary and nuclear boundary.P6 and RD21 co-infiltrated N.Benthamiana,observation found that P6 was located inside the cell and was scattered and scattered,and RD21 and P6 co-localization trajectories were at cell boundary boundary,indicating that RD21 can affect the positioning of P6,and P6 does not affect the positioning of RD21.4 Interaction between FvRD21 and SVBV P6Bi FC verified that FvRD21 interacts with P6.It was found that the areas where YFPⁿ-P6+YFP^c-RD21 and YFPⁿ-RD21+YFP^c-P6 respectively co-infiltrated produced yellow fluorescence,and the yellow fluorescence intensity was similar,indicating that the precursor protease form RD21 can interact with P6 in vivo.The areas where YFPn-P6+YFPc-i RD21 and YFPn-i RD21+YFPc-P6 respectively co-infiltrated produced yellow fluorescence,and the yellow fluorescence intensity was weak,indicating that the active protease intermediate form i RD21 can interact with P6 in vivo.LCI verified that FvRD21 interacts with P6.n Luc-P6 and c Luc-RD21 co-infiltrate N.Benthamiana,spraying the reaction substrate fluorescein,the plant molecular imaging system observation found that n Luc-P6+c Luc-RD21 produces fluorescence,and the interaction fluorescence intensity was similar to that of the positive control group,proving that RD21 interacts with P6 in vivo.5 SVBV infection inhibits ABA metabolism pathway in Fragaria vescaThe genome cloning SVBV was vacuum-filtered to inoculate forest strawberries,and the leaves of the plants showed obvious symptoms of chlorosis and vein embedding.With the extension of the infection time,the transcription level of FvRD21 in the infected strawberry gradually increased.Contrary to FvRD21,the downstream ABA response gene Fv LOG2 gradually decreases,the transcription level of the ABA synthesis gene Fv NCED1,Fv NCED3 first increased and then decreased,and the transcription level of the ABA inhibitor gene Fv PP2C first decreased and then increased.It shows that SVBV infection,the plant produces vein embedding symptoms,and ABA metabolism pathway in the body is inhibited.6 Exogenous ABA treatment induces FvRD21 expressionForest strawberry exogenous 100u M ABA treatment for 1 day,RT-q PCR detection found that FvRD21 transcription level increased,indicating that ABA can induce FvRD21transcription expression.The transcription levels of Fv LOG2,Fv NCED1 and Fv NCED3were significantly increased,and the transcription levels of Fv PP2C were significantly decreased.It was proved that exogenous spraying of ABA could induce ABA metabolism pathways in strawberry plants.7 Exogenous ABA treatment reduces SVBV infection in Fragaria vescaSVBV was inoculated with forest strawberry,and one day later,it was sprayed with ABA and cultured until the disease occurred,SVBV+ABA diseased strawberry plants have more scattered veins and weaker chlorosis,the transcription level of FvRD21 is significantly reduced,and the transcription level of Fv LOG2 and Fv PP2C is significantly increased,the transcription levels of Fv NCED1 and Fv NCED3 were significantly reduced,indicating that exogenous ABA treatment interacts with SVBV to inhibit the response of ABA metabolic pathway.SVBV coat protein P4 and silencing suppressor P6 m RNA transcription levels are reduced,suggesting that exogenous ABA treatment reduced the ability of SVBV to infect strawberry.