The Interaction Between The Eureka Lemon Ribosomal Protein CsRPS9-2 And Citrus Yellow Vein Clearing Virus Coat Protein Affects Viral Infection

Lemon is an economically important fruit in China and the occurrence of diseases can cause serious damage to lemon,of which citrus yellow vein clearing disease is one of the important diseases that cause serious damage to lemon.Viruses in the species citrus yellow vein clearing virus(CYVCV),a member of the genus Mandarivirus in the family Alphaflexiviridae.The CYVCV genome consists of a highly conserved positive-sense RNA of approximately 7.5 kb,with six predicted open reading frames(ORFs).Previous studies have shown that CP plays an important role in virus replication and has been identified as an RNA silencing suppressor(RSS).However,the targets of CP in host have not been identified.In this study,we first constructed a c DNA library of Eureka lemon,and screened candidate reciprocal host factors by yeast two-hybrid system,31 proteins host targets of CP were screened out,which were divided into several types based on their functions,including photosystem I,DNA-binding transcription factor,transferase,chlorophyll-binding protein and chloroplast periplasm.Subsequently,CsRPS9-2,which had the highest occurrence rate,was selected as a candidate factor and the interaction between CP and the candidate factor CsRPS9-2 was verified by validating the interaction in vivo and in vitro.Finally,CsRPS9-2was expressed and silenced in Nicotiana benthamiana and Eureka lemon to investigate the involvement of CsRPS9-2 in host resistance to CYVCV.The main findings are as follows:1、Screening and identification of interacting proteins.Constructed a c DNA library of Eureka lemon.The c DNA library was screened for a total of 31 candidate host factors interacting with the CP protein by yeast two-hybrid assay,using p GBKT7-CP as the bait protein.A yeast two-hybrid was used to validate 31 proteins in photosystem I reaction centre N subunit(PSI-N),non-specific lipid transfer protein 1(ns LTP),40 S ribosomal protein S9-2(RPS9-2),acyl coenzyme A thioesterase 13(ACOT13),40 S ribosomal protein Sa-2(RPSa-2)and the ethylene-responsive transcription factor TOE3(TOE3)interacted with CP.Further experiments using bimolecular fluorescence assay(Bi FC),immunoprecipitation(Co-IP),and fluorophore complementation assay(LCI)demonstrated that CP interacted with CsRPS9-2.2 、 Distribution of CP and CsRPS9-2 and identification of key interaction regions.Subcellular localization clarifies that CP is localized to the nucleus,cell membrane and endoplasmic reticulum,and CsRPS9-2 is localized to the nucleus and cell membrane,CP and CsRPS9-2 co-localized to the cell membrane and nucleus.With the bioinformatics analysis,12 truncated mutants were constructed based on the structural features of CsRPS9-2,and on this basis,using yeast two-hybrid,Bi FC confirmed that 22-324 bp of CsRPS9-2 is both the key region for its interaction with CP and the key to the localization of CsRPS9-2 in the nucleus.3 、 Inter-regulation of CP and CsRPS9-2.RT-q PCR assay revealed that transient expression of CP in N.benthamiana could inhibit the expression of RPS9-2.Application of RT-q PCR and Western blot techniques revealed that transient expression of CsRPS9-2 in N.benthamiana and Eureka lemon could significantly inhibit CP expression.After transient expression of CsRPS9-2 with transgenic N.benthamiana lines 16 c,results from confocal microscopy and Western blot assays showed that CsRPS9-2 expression inhibited the activity of CP as a silencing suppressor activity.4 、CsRPS9-2-mediated resistance.Virus induced gene silencing(VIGS)was used to silence RPS9-2 in N.benthamiana.The RT-q PCR results showed that silencing of RPS9-2reduced the expression levels of SA pathways(NPR1,WRKY40,HSP,PR2,EDS1,PR1 and TGA),HR markers(R-gene,UEP1 and HIN1)resistance genes and photosynthesis-related(Psb O and ABC10)genes were reduced,but the expression levels of chlorophyll biosynthesis-related genes(HEMA1 and CAO)were up-regulated,negatively regulating CP replication in plants.Transient expression of CsRPS9-2 using the plant expression vector p Nm GFPer in N.benthamiana and Eureka lemon increased in vivo plant expression of SA pathways(NPR1,WRKY40,HSP,PR2,EDS1,PR1 and TGA),HR marker(R-gene,UEP1 and HIN1)resistance genes and photosynthesis-related(Psb O and ABC10)base expression levels,resulting in reduced expression levels of chlorophyll biosynthesis-related genes(HEMA1 and CAO).5、Evaluation of resistance in CsRPS9-2 transgenic Eureka lemon.Thirteen transgenic Eureka lemon plants overexpressing CsRPS9-2 were successfully obtained by Agrobacterium-mediated genetic transformation.Analysis by RT-q PCR revealed that the level of CYVCV in CsRPS9-2 transgenic Eureka lemon plants was approximately 50% of that in wild-type plants 1 month after inoculation and that mild yellowing and vein clearing symptoms were only observed in the transgenic plants 4 months after inoculation.The results of electron microscopic analysis also showed slight deformation of chloroplasts in CYVCV-infected transgenic Eureka lemon plants compared to WT Eureka lemon.