Characterization Of The Interaction Between Proteins Of Citrus Yellow Vein Clearing Virus And Identification Of The RNA Silencing Suppressor

Citrus yellow vein clearing virus(CYVCV),belonging to the genus Mandarivirus of the family Alphaflexiviridae,is a newly identified virus,which caused severe diseases in some citrus species.CYVCV virions are a flexuous filament of about 650 nm in size and contain a plus-sense single-stranded genomic RNA of 7.5 kb,consisted of six open reading frames(ORFs).ORF1 encodes an RNA-dependent RNA polymerase(RdRp).ORFs 2-4 compose a triple gene block(TGB)and encode three movement-related proteins.ORF5 encodes a coat protein(CP)and ORF6 encodes a 23 kDa protein with unknown function.In this study,interactions between each pair of five CYVCV proteins were investigated by yeast-two-hybrid(Y2H)assay in-vitro combined with bimolecular fluorescence complementation(BiFC)in planta.Their subcellular localizations were visualized in N.benthamiana leaf cells.Meanwhile,one RNA silencing suppressor(VSRs)encoded by the virus was identified.Results are listed as follows:1.The sequence characterization of five ORFs of CYVCV-HB isolate were identical with other reported isolates.Nucleotide similarity ranges between 96.5-97.1%for TGBpl,99.4-99.7%for TGBp2,97.8-98.9%for TGBp3,96.8-98.6%for CP and 98.1-99.0%for 23K.However,triple gene block encoded proteins,TGBpl,2 and 3 shared amino acid similarity ranges of 97.8-99.1%,98.1-100%and 96.7-100%respectively.Amino acid sequence alignment of coat protein was 96.6-98.5%identical where as 23K was 97.3-98.2%.2.The self-and heterogonous interaction of each pair of proteins were investigate by y2H.CYVCV CP,TGBpl and TGBp2 showed self-interaction whereas CP can interact with each of the TGB proteins,and TGBpl can interact with TGBp2.No interaction was detected for the remaining single or pairs of proteins.To further confirm the interactions identified in the Y2H assays,BiFC assays were conducted in N.benthamiana leaves.Fluorescent signals were observed in the leaf cells that were co-expressed of paired genes including TGBpl-TGBp2 and CP with each TGBp.It was noticed that the subcellular localization of fluorescent signals produced by these combinations differed substantially.The CP,TGBp1 and TGBp2 self-interaction emerged as numerous granular structures in the cytoplasm.The interactions between CP with TGBp1 produced discontinuous punctate spots along with the cell membrane.The interaction of TGBp1 with TGBp2 andCP with TGBp2 produced continuous or intermittent fluorescent signals in cell periphery.Inconsistent with Y2H tests,there were no interaction detected in any of the other combinations.3.Subcellular localization analysis in Nicotiana benthamiana leaves revealed that CYVCV CP and TGBps showed specific localizations when individually expressed.Of those,diffused fluorescent signal of TGBp1 was observed as punctate spots along with the cell membrane and at nuclear periplasm.The fluorescence signals of TGBp2 was observed overspreading on ER-like structures,accumulated as granular-like structures with different sizes on the ER and a sizeable mass near the nucleus Also,TGBp2 co-expressed together with an mCherry-HDEL protein marker showed a partial colocalization pattern on the ER membrane network.Whereas,the TGBp3 fluorescent appeared as discrete granular structures in cytoplasm near CW.The fluorescence of CP was observed as punctate spots along with the cell membrane,occasionally into the nucleus periphery apart from the nucleoplasm.Additionally,vesicles,mostly formed by CP,were also observed in perinuclear and the plasma membrane region.When green fluorescence signals were captured at lower layers of leaf cells,the fluorescence was also observed in endoplasmic reticulum(ER).To further confirm the localization of CYVCY CP on ER,CP was co-infiltrated with mCherry-HDEL marker protein,the fluorescence released by CP was observed well overlapping with the fluorescence released by mCherry-HDEL.4.The co-localization features of these CYVCV proteins reveled that subcellular positions of these proteins were changed under the co-expression situation as compared with those observed for individual expression.Subcellular assay concluded that when CYVCV CP and TGBps co-expressed,they gathered inside plasmodesmata(PD)along with cell wall functioning in cell-to-cell movement through PD,and on endoplasmic reticulum(ER)membranes,most likely involved in forming viroplasm-like complexes as viral factories for viral replication and assembling.5.Moreover,five open reading frames(ORFs)of CYVCV were screened for their local and systemic suppressor activities.Results direct that Coat protein ORF5 showed strong local RNA silencing suppressor activity.None of the proteins analyzed could block the systemic GFP-RNA silencing signals.These findings may contribute to the development of novel genetic resistance against citrus yellow vein clearing virus and related plant viruses.