Suppression Of Post-transcriptional Gene Silencing By The V2 Protein Of Mulberry Mosaic Dwarf-Associated Geminivirus

Geminiviruses are a group of circular single-stranded DNA viruses which occur worldwide.Geminiviruses have caused huge economic losses to crops including cotton,cassava,tomato,and tobacco in many countries.As intracellular obligate parasites,geminiviruses absolutely depend on the host machinery for their replication and movement.To defend themselves against invading geminiviruses,plants have evolved multiple layers of plant defense responses.RNA silencing is a conserved sequencespecific gene regulation process that has been shown to be one of the main anti-geminiviral defenses in plants.In the process of the evolution with plants,geminiviruses have evolved corresponding countermeasures.For example,geminiviruses encode proteins,that is,RNA silencing suppressors,to inhibit RNA silencing-based defensive response.Mulberry mosaic dwarf-associated geminivirus(MMDaV)is a monopartite geminivirus which causes mosaic and dwarfing symptoms in Chinese mulberry trees.MMDaV encodes five open reading frames(ORFs,V1,V2,V3,V4,and V5)on the virion-sense strand,and two ORFs(C1 and C2)on the complementary-sense strand.But the functions of its encoded proteins are unknown.In this study,we identified the potential RNA silencing suppressor of MMDaV,and analyzed the mode of action and subcellular localization of the RNA silencing suppressor.Sequences encoding the seven full-length ORFs were individually cloned into the pCHF3 vector under the control of a 35 S promoter.A mixture of Agrobacterium containing 35S-GFP and plasmids expressing individual ORFs of MMDaV was co-infiltrated into Nicotiana benthamiana GFP line 16 c plants.We found that leaves infiltrated with 35S-GFP and V2 showed a strong GFP fluorescence in the infiltrated area at 5 days post inoculation,suggesting that MMDaV V2 is a potent suppressor of local RNA silencing.GFP fluorescence was not observed in co-infiltrations that contained 35S-GFP,35 SdsGFP,and V2,suggesting that MMDaV V2 does not suppress double-stranded RNA-induced RNA silencing.To further identify critical motifs involved in the silencing suppression activity of V2,we analyzed the sequences of V2 and found that V2 contains an F-box like motif.Mutagenesis analysis showed that the F-box motif was required for V2 to suppress PTGS.Confocal laser microscopy showed that V2 localized to both subnuclear foci and cytoplasm,while V2 mutant appeared not to form discrete foci in the plant nuclear,indicating that F-box is necessary for V2 to localize in the nucleoli.In order to screen the host factor that is involved in the inhibition of PTGS by V2,the interaction between V2 and its candidate protein NbSkp1.1 was verified by yeast two-hybrid and bimolecular fluorescence complementation(BiFC)assay.Virus-induced gene silencing of NbSkp1.1 showed that silencing of NbSkp1.1 impaired the silencing suppression activity of V2.Moreover,the mutation of Fbox changed the V2-NbSkp1.1 interaction sites from the nucleus and cytoplasm mainly to the nucleus.Finally,we analyzed the intracellular localization of the V2 protein of MMDaV.We showed that the V2 protein colocalized with the nucleolar protein fibrillarin(NbFib2)in the nucleolus upon transient expression in the epidermal cells of Nicotiana benthamiana.A yeast-two hybrid assay,followed by BiFC assays,demonstrated the specific interaction between V2 and NbFib2.And we found that the presence of MMDaV excluded the V2 protein from the nucleolus to nucleoplasm.We also presented evidence that the replication-associated protein A(RepA)protein of MMDaV interacted with V2 and enabled the nucleolar exclusion of V2 and the site of V2-V2 interaction.We further revealed that RepA promoted V2 out of the nucleolus presumably by directing the NbFib2-V2 complex from the nucleolus to the nucleoplasm.