Importin/exportin-mediated nucleocytoplasmic shuttling of cucumber mosaic virus 2b protein is required for 2b’s efficient suppression of RNA silencing

Fig 1. 2b has an NES motif for translocation from nucleus to cytoplasm. (A-E) Effect of a mutation in NES on the subcellular localization of 2b in N. benthamiana. The GFP-tagged wild-type 2b (2b-GFP) or L77/85/87A was co-expressed with Ds-Red-fused histone 2B (H2B-DsRed), a nuclear marker protein, in N. benthamiana by agroinfiltration. LMB (40 nM) was infiltrated into the leaves at 2 days post agroinfiltration. (A) GFP fluorescence was observed with an epifluorescence microscope at 4 h after the LMB treatment. Scale bar: 50 μm (white), 5 μm (yellow). (B) Western blot of nuclear-rich fractions to detect 2b-GFP. The numbers above the blot are 5 replicates, and the identical numbers below 2b-GFP and L77/85/87A-GFP indicate the same leaves were used. The histone protein (H3) was detected using anti-H3 antibodies as a loading control. (C) Relative 2b-GFP/H3 ratios were densitometrically calculated using Multi Gauge Software (Fujifilm) and shown as a fold change. Mean values (±SE) were tested on log-transformed data for a significant difference using Student’s t-test (**P < 0.01). (D) Western blot analysis using the nuclear-rich fractions to detect 2b-GFP from the LMB-treated leaves. The…

Fig 2. Mapping of phosphorylation sites of 2b. (A) Schematic structures of the 2b mutants expressed in E. coli and in vitro phosphorylation assay. White boxes: Y2b peptide sequence; gray boxes: HL2b peptide sequence. Black bars: serine (S) or threonine (T); red bars: alanine. When the mutant 2b backbone is the construct containing alanine-substituted residues on all the putative phosphorylation sites, the mutant names contain “[A]”. Purified mutant 2b proteins were co-incubated with (+) or without (-) PKC. Phosphorylated proteins were detected by phosphoprotein gel staining (upper panel); the same gel was stained with Coomassie brilliant blue (lower panel). (B) In vivo phosphorylation assay. White boxes: N-terminal half of Y2b; gray boxes: C-terminal half of HL2b. Black bars: putative phosphorylation residues (S, T or Y); red bars: alanine-substituted residues. Fraction numbers are above the lanes. Elution 1–5: Elution fraction 1–5. Arrows on right: phosphorylated 2bs.

Fig 3. Effect of S28 phosphorylation on subcellular localization of 2b. (A) Subcellular localization of 2b and the 2b mutants in N. benthamiana cells. 2b-GFP and the GFP-tagged 2b mutants (S28A-GFP and S28E-GFP) were co-expressed with H2B-DsRed in agroinfiltrated leaves. GFP fluorescence was observed at 2 days post agroinfiltration using a confocal microscope. Scale bar: 50 μm (white) or 5 μm (yellow). (B) Detection of 2b and S28A in the nuclear-rich fraction. 2b-GFP and S28A-GFP were overexpressed in agroinfiltrated leaves, and the nuclear-rich fraction was probed for the proteins in a western blot. The GFP-tagged proteins and H3 (the loading control) were detected using anti-GFP antibodies and anti-H3 antibodies, respectively. The numbers above the blot are 5 replicate leaves; identical numbers indicate the same leaves were used for 2b-GFP and S28A-GFP. (C) Relative 2b accumulation levels. The relative 2b-GFP/H3 ratios were densitometrically calculated using Multi Gauge Software and shown as a fold-change. Mean values (±SE) were tested on log-transformed data for a significant difference using Student’s t-test (***P < 0.001). (D) In vivo co-immunoprecipitation of NbIMPα1 and 2b-GFP.…

Fig 4. RSS activity of 2bs with mutations at phosphorylation sites. (A) Schematic structure of expression vector constructs containing mutant 2bs. The mutant 2bs were created by substituting S28, S40 and S42 with alanine (A), glutamic acid (E) or aspartic acid (D). (B) RSS activity of the mutant 2bs in the agroinfiltration transient expression assay. Agrobacterium cultures carried a 5:1:5 ratio of the GFP-sense (GFP), GFP-inverted-repeat (GFP-IR), and the 2b constructs. N. benthamiana leaves were agroinfiltrated with bacterial suspensions (~1.0 OD600). GFP was examined at 2 days post-infiltration. Inoculum containing GFP and GFP-IR was used as a control for GFP silencing. (C–E) Comparison of RSS activity between 2b and S28A in N. benthamiana. The Agrobacterium suspension of 2b or S28A was serially diluted to OD600 = 0.5, 0.25 and 0.13. The inocula were a mixture of Agrobacterium cultures carrying the GFP, GFP-IR and the 2b constructs. (C) Leaves were examined for GFP fluorescence at 2 days post-infiltration. (D) Real-time RT-PCR to compare GFP mRNA levels between 2b and S28A. The agroinfiltrated patches, where GFP, GFP-IR and 2b were simultaneously expressed, were excised. Mean values…

Fig 5. Effect of alanine substitution at S28 on CMV disease severity. (A) CMV symptoms on N. benthamiana 10 days post inoculation (dpi) with CMV containing S28A at early stage of infection. Wild-type 2b and S28A genes were inserted into the CMV-H1 vector (H1:2b and H1:S28A). CMV RNA levels based on real-time RT-PCR in inoculated (B) and systemically infected (Systemic) leaves (C). Means (±SE) are fold-changes, calculated when the value of H1:2b is set to 1.0, and tested on log-transformed data for a significant difference using Student’s t-test (*P < 0.05 or **P < 0.01). (D) Disease severity induced by 2b and 2b-S28A and (E) CMV RNA levels based on real-time qRT-PCR in systemically infected upper leaves at 21 dpi. Mean values (±SE) are fold-changes and statistically analyzed as above (****P < 0.0001).

Fig 6. NES motif determines 2b’s RSS activity and CMV accumulation. (A) Alignment of 2b amino acid sequences belonging to subgroup I and II. Amino acid positions are indicated above the alignment. Red arrows indicate leucine residues important for 2b nuclear export. (B) RSS activity in 2b and NES mutant after infiltration of N. benthamiana leaves with Agrobacterium suspensions carrying GFP, GFP-IR and 2b at a ratio of 5:1:5. GFP florescence was observed at 2 days post-infiltration. (C) Western blot analysis to compare the GFP levels. GFP in the agroinfiltrated patches was detected by western blot analysis using anti-GFP antibodies. RuBisCo large subunit is shown as a loading control. The ratios of GFP/RuBisCo were densitometrically calculated when the value in GUS was set to 1.0. (D–G) Effect of a mutation in the NES motif on (D) CMV cyclic symptoms and (E–G) viral accumulation at 10, 15 and 21 dpi. N. benthamiana plants were inoculated with CMV containing either the wild-type 2b or the NES mutant (L77/85/87A). Viral RNA accumulation levels were detected by real-time RT-PCR. Means (±SE) are fold-changes calculated when the value in H1:2b was set to 1.0 and were tested on log-transformed…

Fig 7. Comparison of levels of phosphorylated 2b in nucleus. (A) Western blot analysis to detect nuclear-localized 2b-FLAG protein in the nuclear-rich fraction from leaves of N. benthamiana expressing the FLAG-tagged 2b and L77/85/87A. Histone H3 was detected using anti-H3 antibodies for a loading control. Values below the blot for 2b are the relative ratio of 2b/H3. (B) Detection of cytoplasmic 2b by immunoprecipitation. The cytoplasmic fractions were separated from the total plant extracts by differentiated centrifugation. The 2b-FLAG was immunoprecipitated with anti-2b antibodies and detected in the western blot using anti-FLAG antibody. (C) Detection of phosphorylated 2b in the nuclear fraction. The level of phosphorylated 2b-FLAG in the nuclear-rich fraction was isolated using a phospho-protein enrichment column. Phosphorylated 2b in each elution (E1–5) was detected by western blot using anti-FLAG antibody.

Fig 8. Summary of possible interactions between 2b and players in the RNA silencing pathway in cells. Subcellular trafficking of 2b and its operation points to suppress RNA silencing are shown as red bold arrows. Cytoplasmic 2b is first phosphorylated by a host kinase. The phosphorylated 2b can interact with IMPα, and the 2b/IMPα complex passes through the nuclear pore complex (NPC) into the nucleus and possibly into the nucleolus. AGOs are also imported into the nucleus and nucleolus in a similar IMP-mediated manner. 2b may be recycled after dephosphorylation by host phosphatase(s) in the nucleus or nucleolus; dephosphorylated 2bs will be exported to the cytoplasm in a XPOI-dependent manner, while they can interact with AGOs and sRNAs either in nucleus or nucleolus [37]. AGOs are also exported to the cytoplasm in a XPOI-dependent manner, transporting mature miRNAs to the cytoplasm [39]. Therefore, 2bs seem to shuttle between the nucleus and the cytoplasm as they efficiently track targets such as AGOs and siRNAs.