The subcellular distribution of Cucumber mosaic virus LS2b protein, correlation between its nuclear localization and predicted nuclear localization signals

Cucumber mosaic virus (CMV) 2b protein, a gene silencing suppressor, shows cytoplasmic localization and predominantly nuclear localization due to its two nuclear localization signals (NLSs) regions. We used a series of fluorescent organelle markers and two cytoskeleton markers co-localized with GFP tagged Fny2b (the 2b protein encoded by the CMV Fny strain) fusion protein transiently expressed in leaf epidermal cells of Nicotiana benthamiana by agroinfiltration. Subcellular localization of Fny2b on microtubules was confirmed by perfectly merging with mCherry-MAP (Microtubule-associated protein) marker. High consistency of de-polymerization effect on GFP-Fny2b and microtubules after oryzalin treatment imaged by confocal scanning microscopy was an evidence of Fny2b microtubule localization. Western blot analysis showed that drug treatment affected solubility characteristics of Fny2b, however, oryzalin treatment did not affect its gene silencing suppression function. We need further research to investigate the effect of 2b protein subcellular distribution on its function. We also investigated the nuclear localization ability of transiently expressed GFP-GUS protein fused with various strains of 2b protein. Nuclear localization ability analysis of each 2b protein was obtained in this paper, which is expected to provide an experimental basis to further analyze the correlation between nuclear localization and its predicted nuclear localization signals function. Finally, we successfully verified the great capability of PGsNLS oligo nucleotides as an effective nuclear localization signal could localize macromolecular protein in nucleus.