The rubisco small subunit is involved in tobamovirus movement and Tm-2²-mediated extreme resistance

Abstract

The multifunctional movement protein (MP) of Tomato mosaic tobamovirus (ToMV) is involved in viral cell-to-cell movement, symptom development, and resistance gene recognition. However, it remains to be elucidated how ToMV MP plays such diverse roles in plants. Here, we show that ToMV MP interacts with the Rubisco small subunit (RbCS) of Nicotiana benthamiana in vitro and in vivo. In susceptible N. benthamiana plants, silencing of NbRbCS enabled ToMV to induce necrosis in inoculated leaves, thus enhancing virus local infectivity. However, the development of systemic viral symptoms was delayed. In transgenic N. benthamiana plants harboring Tobacco mosaic virus resistance-2² (Tm-2²), which mediates extreme resistance to ToMV, silencing of NbRbCS compromised Tm-2²-dependent resistance. ToMV was able to establish efficient local infection but was not able to move systemically. These findings suggest that NbRbCS plays a vital role in tobamovirus movement and plant antiviral defenses.

Figure 1.

ToMV MP and NbRbCS interacted in yeast and in vitro. A, Growth of yeast strains containing NLS-LexA BD (control) or NLS-LexA BD ToMV MP baits transformed with AD-NbRbCS or AD (control) on Leu-deficient medium containing Gal and raffinose (Raf; left) or Glu (right) at 28°C for 3 d. Yeast cells were plated at 10- and 100-fold dilutions. B, GST pull-down assay to confirm the interaction between ToMV MP and NbRbCS in vitro. The total soluble proteins of E. coli expressing the NbRbCS-3×FLAG-6×His fusion protein or an empty vector 3×FLAG-6×His were incubated with purified GST-ToMV MP or GST immobilized on glutathione-Sepharose beads. Beads were washed and analyzed by SDS-PAGE and western-blot (WB) assays using anti-FLAG antibody (top panel). The bottom panel shows inputs of purified fusion proteins in pull-down assays. Equal aliquots of glutathione beads loaded with GST-ToMV MP or GST were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Black arrows indicate the bands corresponding to GST-ToMV MP and GST. [See online article for color version of this figure.]

Figure 2.

In vivo pull-down assay for the interaction between ToMV MP and NbRbCS. Immunoprecipitation (IP) was performed on extracts containing transiently expressed 3×HA-NbRbCS and ToMV MP-4×Myc or extracts containing transiently expressed ToMV MP-4×Myc and 3×HA by using protein A/G agarose beads incubated with anti-HA antibody. After immunoprecipitation, precipitates were analyzed by western blot using anti-Myc (top panel) or anti-HA (middle panel) antibody. The expression of ToMV MP-4×Myc was confirmed by western-blot analysis with anti-Myc antibody (bottom panel). IB, Immunoblot.

Figure 3.

Analysis of NbRbCS interaction with ToMV MP deletions. The ToMV MP deletion derivatives used in Y2H assays are depicted in the left diagram. Boxes in the top bar represent the coding region, and the amino acid residues and relative positions in ToMV MP are indicated under each construct. The bars on the bottom indicate the segmental and full-length derivatives used in each assay. For each hybrid, dilutions of yeast cultures at 10⁻¹, 10⁻², and 10⁻³ were spotted onto Leu-deficient (left) or 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal)-containing (right) plates and grown for 3 d at 28°C. SC-UTH, Synthetic complete medium lacking uracil, Trp, and His; SC-UTHL, synthetic complete medium lacking uracil, Trp, His and Leu; RAF, raffinose.

Figure 4.

In vivo subcellular localization of the NbRbCS-ToMV MP interaction by BiFC assay. A to H, NbRbCS and control N-terminal luciferase (Nluc) were tagged at the N terminus with cYFP (cYFP-NbRbCS and cYFP-nLUC) and were coexpressed transiently with ToMV MP or control ToMV P50 tagged at the C terminus with nYFP (ToMV MP-nYFP and ToMV P50-nYFP) in N. benthamiana leaves, and any fluorescence was observed in epidermal (A–D) and mesophyll (E–H) cells by confocal microscopy at 2 dpi. The NbRbCS interaction with ToMV MP was observed in cytoplasmic aggregates (indicated with arrows) and in discrete punctate sites (indicated with asterisks) along the cell edges. I to K, BiFC constructs and the PD marker CFP-tagged TMD (CFP-TMD) were coexpressed in N. benthamiana leaf epidermal cells. YFP channel (I), CFP channel (J), and merged images (K) show that good colocalization existed between the subcellular localization of the ToMV MP-NbRbCS interaction (YFP) and CFP-TMD in discrete punctate sites along the cell border. Green fluorescence shows sites with the two YFP and CFP fluorescence merged. Arrows indicate the positions of overlain YFP and CFP fluorescence. Bars = 20 μm.

Figure 5.

Silencing of NbRbCS induces leaf chlorosis in N. benthamiana plants. A, Western blot (WB) to confirm the silencing of NbRbCS at the protein level. Total protein from equal weights of NbRbCS-silenced leaves and nonsilenced TRV control leaves was separated by SDS-PAGE and stained with Coomassie Brilliant Blue (top panel). Parallel samples were detected by western blotting using anti-RbCS antibodies (bottom panel). For each sample, protein fractionation and immunoblotting were performed with three replications. B, Silencing of NbRbCS resulted in leaf chlorosis. Expanded upper leaves (L) and shoot apices (S) within the dotted rectangles are shown in magnification on the left. Photographs were taken 3 weeks after VIGS infiltration. Bars = 1 cm.

Figure 6.

Silencing of NbRbCS enables ToMV to induce local necrosis. ToMV induced necrosis in the inoculated leaves of NbRbCS-silenced N. benthamiana plants. Leaf photographs were taken at 60 h post infection (hpi) when necrosis appeared on NbRbCS-silenced plants (left column). After being cleared with ethanol, leaves were photographed again (right column). Bars = 1 cm.

Figure 7.

Silencing of NbRbCS delays systemic infection by ToMV. A, Silencing of NbRbCS delays the systemic symptoms induced by ToMV. Plants were photographed at 5 dpi (bottom row). The white arrow points out the shoot curling caused by ToMV systemic infection. Shoot apices are shown in partially enlarged images in the top row. Bars = 1 cm. B, RT-PCR detection of ToMV RNA from TRV control plants with viral symptoms and NbRbCS-silenced plants without viral symptoms at 5 dpi. Representative gels show RT-PCR products (32 cycles of amplification) corresponding to fragments of ToMV CP and the control 18s rRNA. C, Percentage of plants that displayed ToMV-induced systemic symptoms. All values depicted with symbols and bars in line charts are means ± sd from six independent experiments. D, Box plots depict dpi for ToMV systemic infection. Significantly slower ToMV systemic infection was observed in NbRbCS-silenced plants compared with that in nonsilenced TRV control plants. Asterisks above the bars denote the significance of differences between NbRbCS-silenced plants and nonsilenced control plants according to two-sample Student’s t test (**P < 0.01; n = 51). Whiskers represent 5% to 95% intervals of each grouped data set, and crosses represent maximal and minimal observations, respectively.

Figure 8.

Silencing of NbRbCS compromised Tm-2²-mediated local resistance against ToMV. ToMV triggered the HR in inoculated leaves in NbRbCS-silenced but not in nonsilenced control Tm-2² transgenic plants. A, Photographs were taken at 4 d post ToMV infection (left column), and then the excised leaves were decolored in ethanol and photographed again (right column). Red arrows indicate the HR lesions. Bars = 1 cm. B, RT-PCR detection of ToMV CP transcripts in inoculated leaves from NbRbCS-silenced and control Tm-2² transgenic plants. The number of PCR cycles is shown above each lane. β-Actin was included as a loading control, and photographs were typical results from three repeated experiments.