The Subcellular Distribution Of Cucumber Mosaic Virus LS2b Protein,and The Interaction With Endogenous 30S Ribosomal Protein Subunit S11 Affects Viral Replication,Infection And Gene Silencing Suppressor Activity

For its wide distribution,high level replication and uncomplicated typical viral genetic structure,Cucumber mosaic virus(CMV)which has already been long-term studied is an important model plant virus for understanding plant-virus interaction process and mechanism.Multifunctional CMV2 b protein has been identified as a virulence factor and confirmed as an efficient gene silencing suppressor.CMV2b shows cytoplasmic localization and predominantly nuclear localization due to its two NLS regions.We used a series of fluorescent organelle markers and two cytoskeleton markers co-localized with GFP tagged Fny2 b fusion protein agroinfiltration transiently expressed in leaf epidermal cells of Nicotiana benthamiana.Subcellular localization of Fny2 b on microtubules was affirmed by perfectly merging with mCherry-MAP marker.High consistency of de-polymerization effect on GFP-Fny2 b and microtubules after oryzalin treatment imaged by confocal scanning microscopy was an evidence of Fny2 b microtubule localization.To study the correlation between the differences of a nuclear localization signal sequence and the nuclear localization capability of various CMV strains,we amplified the CMV2 b protein gene of various strains,cloned into expressing recombinant plasmids pBI-GFP-GUS.The expression vector contain each seven strains of 2b gene and a mutant were injected into leaf epidermal cells of Nicotiana benthamiana.Nuclear localization ability of each 2b protein observed by confocal scanning microscopy were obtained and analyzed the correlation between nuclear localization and its predicted nuclear localization signals function.We screened Arabidopsis thaliana cDNA library interacted with CMV 2b protein by using yeast two-hybrid system,twelve host proteins were identified as CMV LS2 b binding partners.Among of them,30 S ribosomal protein subunit S11(RPS11)was chosen to be further studied.The RPS11-LS2 b interaction was confirmed by full-length yeast two-hybrid system.We used BIFC assay observed by confocal laser microscope and GST pulldown assay to demonstrate the interaction between endogenous RPS11 and viral CMVLS2 b both in vivo and in vitro.We used TRV-based gene-silencing vector to knockdown NbRPS11 transcription.Immunoblot analysis demonstrated that infectious viral RNA replication declined and CMV infection increased in RPS11 down-regulated Nicotiana benthamiana plants which indicated that knockdown of RPS11 inhibited CMV replication and accumulation.Gene silencing suppressor activity of CMV2 b protein reduced on effect of RPS11 knockdown.Conclusively,all the evidences proved that interaction with host RPS11 protein profoundly affect function of viral protein.