Fig 1.
Sugarcane streak mosaic virus P1 (P1SCSMV) suppresses both local and systemic RNA silencing in Nicotiana benthamiana.
(A). P1SCSMV could suppress the single-strand sense-GFP (ssGFP) induced local RNA silencing with and without the C-terminal of double-strand GFP fragment (dsFP). The p19 from tomato bushy stunt virus was used as positive control, EV means empty vector. At 3 dpi, the leaves were photographed under a UV lamp, and the proteins were detected with the commercial anti-GFP and anti-FLAG antibodies. Black arrow indicates the target proteins. (B). P1SCSMV could suppress the single-strand sense-GFP (ssGFP) induced systemic RNA silencing. The infiltrated leaves were photographed under a UV lamp after 3 dpi and 11 dpi. (C). Statistical analyses of the systemic RNA silencing suppression ability of P1SCSMV.
Fig 2.
P1SCSMV promotes the pathogenicity of the PVX-derived recombinant virus infection in N. benthamiana.
(A). Disease of the PVX-GFP and PVX-P1SCSMV infected N. benthamiana at 12 dpi. bar represents 3 cm. (B). The disease appeared in all parts of the plant, petiole, and culm upon recombinant PVX-P1SCSMV infection at 16 dpi. bar represents 3 cm. (C). The intensity of cell-death in the local leaves were quantified by the relative electrolyte leakage at 8 dpi. “a” and “b” means significant difference between these two columns. (D). Western blot and RT-PCR confirmed the recombinant PVX-GFP and PVX-P1SCSMV infection at 8 dpi. Similarly, commercial anti-GFP and anti-FLAG antibodies, and anti-CPPVX were used for detection of the expressed GFP, P1SCSMV, and CPPVX. The PVX CP coding sequences was used for quantification of the recombinant PVX accumulation. (E). Northern blot detection of viral genomic RNA accumulation in recombinant virus PVX-GFP and PVX-P1SCSMV infected condition at 12 dpi. The ribosome RNAs (rRNA) were stained as loading control.
Fig 3.
P1SCSMV suppresses the expression of IRE1/bZIP60 marker genes in unfolded protein response (UPR) signaling pathway.
(A). PVX-GFP and PVX-P1SCSMV infiltrated leaves were photographed before and after trypan blue staining at 6 dpi. bar represents 2 cm. (B). Western blot confirmed the expression of the CPPVX, GFP, and P1SCSMV using commercial and self-prepared antibodies at 6 dpi. (C). The cell-death intensity of the systemic leaves was quantified by the relative electrolyte leakage at 6 dpi. (D). The relative expression levels of IRE1/bZIP60 UPR marker genes were determined at 6 dpi. Different letters (a, b, and c) mean significant difference between these three columns which represent the mock-treated, PVX-GFP-infected, and PVX-P1SCSMV-infected conditions. (E). The relative expression levels of the marker genes of IRE1/bZIP60 UPR signaling pathway were quantified at 6 dpi. The three columns in each gene represent mock-treated, transient expression of GFP, and transient expression of P1SCSMV, respectively. (F). Determination of the subcellular localization of the P1SCSMV at 3 dpi. The white frame is enlarged and is listed in the bottom channel. bar represents 50 μm.
Fig 4.
P1SCSMV has a C-terminal bipartite nuclear localization signals (NLS).
(A). Constructs Illustration of different GFP-tagged P1SCSMV. Red letters indicate the C-terminal conventional nuclear export signal (NES) peptide and a N-terminal conventional NLS in the mutants P1SCSMV-nls-NES and P1SCSMV-nls-NLS, respectively. Green letters represent the linker between the signal peptide and the start amino acid (aa, Met) of P1SCSMV. The marked numbers indicate the aa positions. Red frames indicate the two NLS. (B). Subcellular localization of different P1SCSMV mutants listed above. Free red fluorescence protein (RFP) was co-expressed with GFP-tagged P1SCSMV and its derivatives. After 2 dpi, leaves were harvested and observed.
Fig 5.
Self-interactions of P1SCSMV and its NLS mutants in vivo and in vitro.
(A). P1SCSMV was found to interact with itself in the yeast two-hybrid assay (Y2H). γb protein of barley stripe mosaic virus (BSMV) served as the positive control for the self-interaction. Top number indicate the dilution folds of the yeast cells plated in different type of nutrient deficient medium. (B). P1SCSMV associated with itself in vivo in the bimolecular fluorescence complementary assay (BiFC). YFP signals were emitted under a 512 nm wave-length laser and depicted as a false-green color. Scale bar, 40 μm. (C). P1SCSMV was found to interact with itself in vivo in co-immunoprecipitation (Co-IP). The plus sign in each lane indicates co-expression of the two proteins. The commercial anti-GFP and anti-FLAG antibodies were used to confirm the presence of the target protein. (D). The effects of the NLS on P1SCSMV to the self-interaction ability were determined. The self-interaction of the P1SCSMV served as the positive control.
Fig 6.
RNA silencing suppression (RSS) activity of P1SCSMV and its NLS-related mutants.
(A). RSS activity of P1SCSMV and its NLS mutants. The leaves were collected at 3 dpi. Black arrow indicates the target protein. The commercial anti-GFP and anti-FLAG antibodies were used to confirm the expression of the target protein. (B). Western blot detected the P1SCSMV-NLS mutants that were not detected in RSS activity assay. (C). RSS activity of P1SCSMV and its compelling-nuclear-importing (P1SCSMV-NLS) and compelling-nuclear-exporting mutant (P1SCSMV-NES). (D). Western blot detection of protein expression of these P1SCSMV mutants.
Fig 7.
Effects on the recombinant PVX pathogenicity and IRE1/bZIP60 UPR signaling pathway suppression ability of P1SCSMV and its NLS-related mutants.
(A). Symptoms of the recombinant virus by cis- heterologous expressing of the P1SCSMV and its NLS-related mutants through PVX at 9 dpi and 14 dpi. The virus-infected systemic leaves at 9 dpi were harvested and subjected to 3,3’-diaminobenzidine tetrahydrochloride (DAB) staining, trypan blue staining, and tissue printing analyses. (B)-(D). Relative intensity of the ROS, cell-death, and virus accumulation were quantified by the ImageJ software. Different letters (a, b, and c, et. al.) mean significant difference between these columns. (E). Northern blot (upper panel) and western blot were used to determine (bottom panel) the viral genomic RNA and P1 mutant’s accumulations in recombinant viruses infected condition at 9 dpi. The antibodies, genomic RNA, and sub-genomic RNA bands are marked on the right side of the images. (F). Relative expression levels of marker genes in IRE1/bZIP60 UPR signaling pathway under recombinant PVX viruses’ infection. Different letters (a, b, and c, et. al.) mean significant difference between these columns as described above.
Fig 8.
Analysis of the relationships between cell-death intensity, and nucleocytoplasmic-shuttling of P1SCSMV, IRE1/bZIP60 UPR signaling pathway, and morphological changes of ER.
(A). The cell-death intensity of the PVX-GFP and PVX-P1SCSMV in leaves that the nuclear importin system was partially inhibited. After 12 dpi of TRV inoculated, PVX-GFP and PVX-P1SCSMV infiltration was performed, and the leaves were collected for trypan blue staining at the following 6 dpi. (B). The cell-death intensity was quantified using the ImageJ software. Different letters (a, b, and c, et. al.) mean significant difference between these columns as described above. (C). The cell-death intensity of the PVX-GFP and PVX-P1SCSMV infected NbbZIP60- and NbBLP4-downregulated N. benthamiana plants (D). Quantification of the cell-death. Different letters (a, b, and c, et. al.) represent the same meaning above. (E). Accumulation levels of the PVX-GFP and PVX-P1SCSMV in N. benthamiana plants that with and without NbImp. α-, NbImp. β, and NbImp. α+β, and NbbZIP60-downregulated condition were determined. The intensity of bands was quantified. The experiment was repeated for independent triple times, and the quantification was performed from independently three images at least. (F) Subcellular localization of the P1SCSMV via overexpressing on the 16C transgenic N. benthamiana at 3 dpi, 4 dpi, and 5 dpi. The RFP monitor the P1SCSMV, and the GFP-HDEL was used to show the ER morphology. Bar scale, 10 μm. White arrow indicates the positions described in the result. White frame was enlarged in last lane (bar scale, 1 μm).
Fig 9.
P1SCSMV inhibited the IRE1-mediated splicing of the NbbZIP60U through directly binding to the stem-loop of NbbZIP60U in vivo.
(A). Phenotype of the N. benthamiana plants with different treatments at 2, 3, and 5 dpi. (B). Illustration of IRE1-mediated splicing of NbbZIP60U (Niben101Scf24096g00018.1) to NbbZIP60S. The start and end of each type of NbbZIP60 were marked and the red arrows represent the pairs of primers designed for the analyses. Red number indicate the lengths of the amplified fragments with the target primers. (C). Quantification of the relative levels of NbbZIP60U and NbbZIP60S in the five-treated N. benthamiana. The levels of PVX CP represents the virus accumulation levels. The experiment was independently repeated for three times, and quantification was performed using at least three independent images. (D). The activation and inhibition of the IRE1/bZIP60 UPR signaling pathway were calculated based on logarithm value of NbbZIP60U/NbbZIP60S in base-10. The upper pink panel represents the UPR activation, and the lower cyan panel indicates UPR inhibition. ** means significant difference with a p-value < 0.01. (E). An RNA-immuno-precipitation assay (RIP) showed the binding of P1SCSMV to the stem-loop of NbbZIP60U through its NLS region in vivo. HC and LC denote “heavy chain” and “Light chain”, respectively. The right panel shows the relative levels of NbbZIP60U and NbbZIP60S using RT-PCR and qRT-PCR analyses of the input and precipitated (IP) samples. In the RT-PCR analyses of IP samples, the PCR cycle was set to 45 for the amplification of the NbbZIP60S and 25 cycles for NbbZIP60U. In the qRT-PCR analyses, the column filled with solid colors represents the amount of bZIP60U isolated from precipitated Flag-GUS, Flag-P1SCSMV, and Flag-P1SCSMV-nls, while the column filled with mosaic colors indicates the amount of bZIP60S isolated from precipitated proteins. Letters (a and b) indicate significant differences between the columns.
Fig 10.
P1SCSMV directly binds to the stem-loop region of NbbZIP60U through via NLS region in vitro.
(A). CBB staining of the purified recombinant GST-tagged GFP, P1SCSMV, and P1SCSMV-nls mutant. Black arrows indicate the target protein. (B). In vitro transcription of the 532-bp 5’-biotinylated stem-loop region containing RNA from NbbZIP60U (NbbZIP60U probe) and its corresponding mutant (NbbZIP60U-M probe). (C). Predicted stem-loop regions located in NbbZIP60U transcripts and their mutants. Blue letters in NbbZIP60U-M probe represent the mutations. (D). In vitro electrophoretic mobility shifts assay was used to analyses the direct binding of GST-P1SCSMV to the stem-loop containing single-stranded RNA by the NLS peptide. The black triangle indicates the gradually increased concentration of the target protein, and the protein concentration gradient of the GST-P1SCSMV and GST-P1SCSMV-nls used were the same (lane 2 & 5, 0.1 μg, lane 3 & 6, 0.5 μg, and lane 4 & 7, 2.5 μg). The labeled probes (NbbZIP60U probe & NbbZIP60U-M probe) used were 1 μL in all lanes. The plus sign represents the lane containing the target protein and corresponding probe. (E). The proposed working model of the P1SCSMV in achieving moderate robust infection. In absence of P1SCSMV, UPR activation leads to increased expression of bZIP60U. bZIP60U then further processed by the ER-localized IRE1 protein to form bZIP60S. Normal translation generated bZIP60S transcription factor will import to the nuclear by the cellular importin α/β system, then mediated expression of stress-related genes and promoted pro-survival of the cell. In UPR activation with P1SCSMV condition, the bZIP60U induced by UPR activation directly bound by the P1SCSMV. The optimal and correct splicing of bZIP60U was inhibited, leading to decreased bZIP60S and bZIP60S protein. All of these decreased the intensity of the bZIP60S-mediated stress-related genes expression, and finally caused PCD of the cell.