Nuclear Exportin 1 (XPO1) Binds to the Nuclear Localization/Export Signal of the Turnip Mosaic Virus NIb to Promote Viral Infection

FIGURE 1. Sequence analysis of the turnip mosaic virus (TuMV) NIb protein. (A) Predicted TuMV NIb nuclear export signal (NES) and nuclear import signal (NLS) motifs are indicated by blue boxes and pink boxes in the NIb sequence. The RNA-dependent RNA polymerase (RdRP) domain is highlighted by the yellow box. (B) Detailed sequence information of the putative NIb NES and NLS motifs. For the mutation of NESs, mNESb or mNESc, tryptophan (Trp, W) was instead of Φ (L,I,V,M, or F) (These mutations are indicated in red). For the mNLSc, the three basic amino acids lysine (K) were replaced with alanine (A) (red).

FIGURE 2. Analyses of the putative NESs of NIb. (A,B) Localization of the four putative NIb NESs. Four predicted sequences of the NIb NES (NESa, NESb, NESc, and NESd) domains and the mutated NESb (mNESb) and NESc (mNESc) sequences (Figure 1B) were fused to the YFP tagged vector. Agrobacterium cultures carrying YFP (as a control), NESa-YFP, NESb-YFP, NESc-YFP, NESd-YFP, mNESb-YFP or mNESc-YFP and NIb-YFP were infiltrated onto RFP-H2B (as a nuclear marker, red) transgenic Nicotiana benthamiana plants, and fluorescence (green) was visualized at 32 h post infiltration (hpi). YFP and NIb-YFP were distributed in both the nucleus and cytoplasm, and the putative NESa and NESd that fused with YFP had subcellular localizations similar to YFP. The putative NESb and NESc domains that fused YFP were able to export YFP from the nucleus to the cytoplasm, but the mutated NESb and NESc domains failed to function in nuclear export. (C) Localization of NIb carrying the NESb mutation (NIb-mNESb), NESc mutation (NIb-mNESc), or NESb and NESc (NIb-mNESb/c). These full-length NIb mutants were fused to the YFP tagged vector and fluorescence (green) was examined at 32 hpi. The single NES mutation on NIb-YFP failed…

FIGURE 3. Analyses of the putative NLSs of NIb. (A,B) Localization of the putative NIb NLSs. Four putative NIb NLS (NLSa, NLSb, NLSc and NLSd) sequences and the mutated NLSc (mNLSc) were fused to the GUS-YFP vector. Agrobacterium cultures carrying the GUS-YFP (as a control), Green fluorescence shows the localization of each protein, and fluorescence was examined at 32 hpi. (C) Localization of NIb and the NIb carrying NLS mutation (NIb-mNLS) fused to GUS-YFP fluorescence at 32 hpi. RFP-H2B as a nucleus marker (red). Images represent single plain micrographs and bars = 25 μm (A–C).

FIGURE 4. NbXPO1a interacts with NIb NES and NLS domains. (A) Y2H assays to detect interactions between NbXPO1a, the a1 and a4 fragments of NbXPO1a with the NIb NES and NLS domains and their respective mutant sequences. NbXPO1a-a1 and NbXPO1a-a4 are two NbXPO1a deletion mutants that contain the IBN_N domain and the CRM1_C domain, respectively. Y2H Gold yeast cells harboring the indicated plasmids were co-expressed, subjected to 10-fold serial dilutions and plated on synthetic dextrose (SD)/-Trp, -Leu, -His, -Ade agar to identify protein interactions. (B,C) BiFC assays in RFP-H2B transgenic N. benthamiana (red) leaves at 32 hpi. Green fluorescence was observed as a consequence of the complementation of YN tagged NbXPO1a, and the a1 (NbXPO1a-a1) and a4 (NbXPO1a-a4) fragments of NbXPO1a with the YC tagged NIb NES or NLS sequences and their respective mutants shown in Figure 1B. The yellow fluorescence results from overlapping of the green BiFC and the red RFP-H2B fluorescence. Bars = 25 μm.

FIGURE 5. The mutation of NESs and NLS of NIb impairs viral infection. (A) The confirmed NESs and NLS in this study were renamed and noted by solid blue boxes and solid pink box, respectively. RdRP, RNA-dependent RNA polymerase, was noted by yellow box. (B–D) The wild type (wt) TuMV-GFP infection clone, and the TuMV-GFP infection clone harboring the mutation of functional NES1, NES2, NES1 and NES2 (NES1/NES2), or NLS were infiltrated onto N. benthamiana leaves. At 60 hpi and 7 days post infiltration (dpi), total RNA and protein were extracted from the infiltrated leaves and systemic leaves for RT-qPCR analysis and western blot analysis (B–D). Values represent means relative to the leaves infiltrated with TuMV plus Mock. Each average value was calculated by three representative replicates. The data were analyzed using Student’s t test and double asterisks indicate P < 0.01. NbActin was used as an internal control. The large Rubisco subunit (rbcL) staining with Ponceau S showed the equal loading. (E) Viral symptoms and GFP fluorescence in plants inoculated with the indicated infectious clones were photographed under natural and UV light at 7 dpi.

FIGURE 6. Overexpression of the IBN_Nand CRM1_C domain of NbXPO1a facilitate viral accumulation and systemic infection. (A) Schematic representation of full-length NbXPO1a and four NbXPO1a fragments. (B) Transiently expressed YFP fusions with full-length NbXPO1a-YFP (FL), NbXPO1a-a1-YFP (a1), NbXPO1a-a2-YFP (a2), NbXPO1a-a3-YFP (a3), or NbXPO1a-a4-YFP (a4) in N. benthamiana leaf cells infected with TuMV-6K2-mCherry-CFP-NIb at 70 hpi. Images represent single plain micrographs and bars = 10 μm. The region of nucleus is highlighted with a white circle. (C) RT-qPCR analyzed TuMV RNA levels. RNA was extracted from the infiltrated leaves as indicated in panel (A). Values represent means relative to the leaves infiltrated with TuMV-6K2-mCherry-CFP-NIb plus Mock. And each average value was calculated by three representative replicates. The data were analyzed using Student’s t test and double asterisks indicate P < 0.01. NbActin was used as an internal control. (D) Viral symptoms of TuMV-6K2-mCherry-CFP-NIb-infected N. benthamiana plants at 16 dpi. Transient overexpression of the full-length NbXPO1a and truncated NbXPO1a fragments in leaf cells infected with TuMV-6K2-mCherry-CFP-NIb. Bars = 2 cm.…