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. 2018 Dec 11;179(2):507–518. doi: 10.1104/pp.18.01342

A Host ER Fusogen Is Recruited by Turnip Mosaic Virus for Maturation of Viral Replication Vesicles1

Nooshin Movahed a, Jiaqi Sun a, Hojatollah Vali b,c, Jean-François Laliberté d, Huanquan Zheng a,2,3
PMCID: PMC6426418  PMID: 30538165

Interacting with 6K2 of TuMV ER fusogen RHD3 is recruited by TuMV for the efficient formation of replication-competent vesicles in infected host cells.

Abstract

Like other positive-strand RNA viruses, the Turnip mosaic virus (TuMV) infection leads to the formation of viral vesicles at the endoplasmic reticulum (ER). Once released from the ER, the viral vesicles mature intracellularly and then move intercellularly. While it is known that the membrane-associated viral protein 6K2 plays a role in the process, the contribution of host proteins has been poorly defined. In this article, we show that 6K2 interacts with RHD3, an ER fusogen required for efficient ER fusion. When RHD3 is mutated, a delay in the development of TuMV infection is observed. We found that the replication of TuMV and the cell-to-cell movement of its replication vesicles are impaired in rhd3. This defect can be tracked to a delayed maturation of the viral vesicles from the replication incompetent to the competent state. Furthermore, 6K2 can relocate RHD3 from the ER to viral vesicles. However, a Golgi-localized mutated 6K2GV is unable to interact and relocate RHD3 to viral vesicles. We conclude that the maturation of TuMV replication vesicles requires RHD3 for efficient viral replication and movement.

ROOT HAIR DEFECTIVE 3 (RHD3) is a plant member of the dynamin-like atlastin GTPase family. Like its mammalian counterpart Atlastin-1 (Hu et al., 2009; Orso et al., 2009) and yeast counterpart Sey1p (Yan et al., 2015), RHD3 plays an important role in the generation of the interconnected tubular ER network (Chen et al., 2011). Localized to the endoplasmic reticulum (ER; Chen et al., 2011), RHD3 is required for efficient homotypic fusion of different ER membranes (Hu et al., 2003; Zhang et al., 2013). All atlastin proteins have a conserved domain structure: an N-terminal GTPase domain, a three-helix bundle-rich middle domain, two transmembrane domains, and a short amphipathic helix C-terminus (Bian et al., 2011; Yan et al., 2015; Sun and Zheng, 2018). It has been suggested that different RHD3 molecules in different ER membranes dimerize through their GTPase and middle domains to tether the ER membranes (Sun and Zheng, 2018). GTP hydrolysis then triggers a conformational change of the RHD3 dimer that eventually brings two different ER membranes together (Sun and Zheng, 2018).

To establish a successful infection, many plant RNA viruses remodel host cellular membranes to create a viral replication organelle that facilitates replication and intracellular movement of the viral RNAs (Laliberté and Zheng, 2014). Turnip mosaic virus (TuMV), a positive-strand RNA virus of the genus Potyvirus (Cotton et al., 2009), remodels the ER to generate viral vesicles. These viral vesicles have been found to be the site for viral genomic replication (Cotton et al., 2009) and serve as a vehicle for intracellular (Grangeon et al., 2012) and the cell-to-cell spread of the viral RNAs (Grangeon et al., 2013). These viral replication vesicles are generated in the ER and are released at the ER exit sites (Wei and Wang, 2008; Jiang et al., 2015). They are believed to take a Golgi by-pass unconventional pathway (Cabanillas et al., 2018) that requires microfilaments to reach plasmodesmata (PD; Cotton et al., 2009) where they ultimately move into uninfected neighboring cells (Grangeon et al., 2013). These viral vesicles generated early in the infection process are usually convolutional membrane structures (CM) still linked to the ER (Wan et al., 2015a). These CMs are replication-incompetent. They bud off and mature into replication-competent single-membrane vesicles (SMVs). In the later infection stage, SMVs mature into larger, double-membrane vesicles (DMVs) and multivesicular bodies (MVBs) for efficient viral movement (Wan et al., 2015a). The transmembrane protein 6K2, encoded by the TuMV genome, plays an important role in the ER membrane reorganization that leads to the formation and maturation of viral replication vesicles (Cotton et al., 2009). However, the contribution of host proteins in the biogenesis and maturation of viral replication vesicles needs to be better defined. It is known that certain host endomembrane factors are involved in the formation of viral replication vesicles. For example, the COPII protein SEC24A and COPI-forming small GTPase ARF1, factors that act in the early secretory pathway, have been shown to be involved in the formation of TuMV and Red clover necrotic mosaic virus replication vesicles, respectively (Hyodo et al., 2013; Jiang et al., 2015). SYP71, an ER-localized SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor), was shown to be involved in the fusion of TuMV-induced vesicles with chloroplasts (Wei et al., 2013). Endosomal sorting complexes required for transport (ESCRT), which are required for the formation of MVBs (Schmidt and Teis, 2012), are involved in membrane curvature and viral replication factory assembly of the Tomato bushy stunt virus and the Brome mosaic virus (Barajas et al., 2009, 2014; Diaz et al., 2015). Finally, synaptotagmin A (SYTA), a SNARE partner involved in the membrane contact between the ER and plasma membrane (Uchiyama et al., 2014), was shown to be recruited to PD for cell-to-cell movement of TuMV, Turnip vein clearing virus, and Cabbage leaf curl virus during infection (Levy et al., 2015).

In this study, we describe a different role of RHD3 in the maturation of replication vesicles of TuMV in infected cells. We observed a considerable delay in the development of the TuMV infection in the Arabidopsis (Arabidopsis thaliana) rhd3 mutant. Immunoblot analyses using anticoat protein confirmed that there is a reduction in viral replication in primary infected cells as well as a delay in the intercellular movement of TuMV to upper leaves in rhd3 plants. Fluorescence microscopy of 6K2 vesicles indicated that the cell-to-cell movement of 6K2 tagged viral vesicles is impaired. Using fluorescence and transmission electron microscopy, we studied the cellular basis of the delay in TuMV infection in rhd3. We observed that RHD3 is required for the formation and maturation of the CM into small SMVs and larger DMVs in the infected cells. Furthermore, we found that 6K2 physically interacts with RHD3, and redirects RHD3 from the ER to viral replication vesicles. These results suggest that during the TuMV infection, RHD3 is recruited by TuMV for the formation and maturation of the replication vesicles for efficient replication and movement of the virus.

RESULTS

Replication and Systemic Movement of TuMV Are Impaired in rhd3-8

Initially, the TuMV replication vesicles are generated as CMs in the ER. They then mature into SMVs, DMVs, and MVBs and move intracellularly through a Golgi bypass route (Wan et al., 2015a; Cabanillas et al., 2018). Some host factors must play a role in the development and maturation of these replication vesicles. We wondered if RHD3, an ER fusogen, played any role in the process. To this end, we checked if there was any infection delay in rhd3-8, a knockout rhd3 mutant allele that has been genetically complemented (Zhang et al., 2013; Sun and Zheng, 2018). Leaves of the wild-type Col-0 and rhd3-8 plants were infected with a 6K2-GFP tagged infectious TuMV clone (TuMV::6K2-GFP) by agro-infiltration. The development of the GFP signal and TuMV symptoms in upper, noninoculated leaves was then monitored. No TuMV infection symptoms were observed in mock-infected plants (Fig. 1A). In the wild-type Col-0 there were a few plants infected by TuMV at 7 d after TuMV::6K2-GFP inoculation but not in rhd3-8 (Fig. 1B). GFP and TuMV symptoms were clearly detected at 9 d postinoculation (dpi) in about 40% of the wild-type Col-0 and but less than 10% of rhd3-8 (Fig. 1B). At 11 dpi, almost 70% of the wild-type Col-0 plants showed GFP and systemic infection in the upper leaves, while less than 30% of rhd3-8 plants had these symptoms (Fig. 1B). At 13 dpi, 95% of the wild-type Col-0 expressed the systemic infection, and only 50% of rhd3-8 plants showed systemic infection (Fig. 1B). At 15 dpi, all the wild-type Col-0 plants were systemically infected, while rhd3-8 plants need 17 dpi to reach 100% systemic infection (Fig. 1B). We concluded that the systematic infection of TuMV in rhd3-8 mutant plants is considerably delayed compared to that in the wild type.

Figure 1.

Figure 1.

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Replication and systemic movement of TuMV are impaired in rhd3-8. A, Symptoms of the wild-type (WT) Col-0 (left in each photo) and rhd3-8 plants (right in each photo) inoculated with mock or TuMV::6K2-GFP. B, Percentage of plants (three repeats of 15, 15, and 20 plants = 50 plants in total) systemically infected with TuMV. Each error bar represents ±se within three repeats. C to F, Immunoblots of extracts from leaves of the mock-infected wild-type Col-0, the wild-type Col-0, and rhd3-8 primarily (C and D); and systemically (E and F) TuMV infected as indicated. Protein loading (D and F) verified by Coomassie staining. G, Quantification of the CP accumulation in primarily and systemically infected cells. The intensity of the bands shown in (C) and (E) was measured using ImageJ.

We wondered if the replication and/or movement of TuMV was affected in rhd3-8. To assess this question, the leaves of the wild-type Col-0 and rhd3-8 were agroinfiltrated with the mock virus or pCambiaTuMV. At 12 dpi, the accumulation of the coat protein (CP) of TuMV was analyzed in primarily and systemically infected leaves (upper noninoculated leaves) of Col-0 and rhd3-8 plants by immunoblot analysis using a rabbit serum against CP (Jiang et al., 2015). The CP level was then quantified using ImageJ (https://imagej.nih.gov/ij/). Generally, in both primarily and systemically infected cells, the CP accumulation in rhd3-8 plants was significantly less than that in the wild-type plants (Fig. 1, C–G). Quantification of the intensity of the bands using ImageJ indicated that, in rhd3-8 plants, there were 77% and 83% reductions in the CP accumulation in the primarily and systemically infected leaves (Fig. 1, C–G), respectively. This result suggested that the mutation in RHD3 affected the replication of TuMV. The more severe reduction of infection in systemically infected leaves (83%) than in primarily infected leaves (77%) also supported the idea that the movement of TuMV is impaired in rhd3-8.

Production and Cell-to-Cell Movement of 6K2-Tagged Vesicles Are Affected in rhd3-8

Next, we examined if the impaired viral replication and systemic movement of TuMV in rhd3-8 mutant plants are related to the production and cell-to-cell movement of 6K2 vesicles. For this purpose, the infectious TuMV::6K2-mCherry/GFP-HDEL construct was expressed either in the wild-type Col-0 or rhd3-8 mutant plants. This dual construct expresses both 6K2-mCherry and GFP-HDEL from the infectious TuMV. The construct allows monitoring of the production of 6K2 vesicles in primarily infected cells (indicated by the presence of both GFP-HDEL and 6K2-mCherry) and in secondarily infected cells (indicated by the presence of 6K2-mCherry only; Agbeci et al., 2013; Jiang et al., 2015; Movahed et al., 2017). As indicated in Figure 2, the production of 6K2-mCherry in primary infection foci, as evidence by the production of green and red fluorescence, was first observed in both Col-0 and rhd3-8 plants after 7 dpi (Fig. 2, A and B). The production of 6K2-mCherry in primary infection foci was then continuously assessed until 15 dpi (Fig. 2, C–F). We found the intensity of 6K2-mCherry in primary infection foci in rhd3-8 cells was significantly weaker than in the wild-type Col-0 cells at all times (Fig. 2G). This finding suggested that the production of 6K2-mCherry-tagged vesicles in the primarily infected rhd3-8 cells is impaired.

Figure 2.

Figure 2.

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Production and intercellular movement of 6K2-mCherry in the wild-type Col-0 and rhd3-8 mutant plants. A to F, Coexpression of the dual construct TuMV:6K2-mCherry/GFP-HDEL in the wild-type Col-0 (A, C, and E) and in rhd3-8 mutant plant cells (B, D, and F) at 7 dpi (A, B); 11 dpi (C, D); and 15 dpi (E and F). Arrows show the intercellular movement of 6K2-mCherry. Scale bars = 10 μm. G, The mean intensity (au/frame) of 6K2-mCherry signals in primarily infected cells indicated by GFP-HDEL in Col-0 (blue) and rhd3-8 mutant plants (red) at the times indicated (n = 20 frames from four different plants of each treatment). H, The percentage of the cells expressing only 6K2-mCherry signal in Col-0 (blue line) and rhd3-8 mutant (red line) plants (n = 150 cells from four different plants of each treatment). I, The mean intensity (au/frame) of 6K2-mCherry signals in secondarily infected cells as indicated by the red-only areas of cells lacking GFP-HDEL (but neighboring those with GFP-HDEL) in Col-0 (blue) and rhd3-8 mutant (red) plants (n = 20 frames from four different plants of each treatment). Each error bar represents ±se. Stars indicate the significant difference found at each time point. Student’s t test, P-value < 0.05.

The intercellular movement of the 6K2-mCherry vesicles in both the wild-type Col-0 and rhd3-8 mutant plants was then assessed by analyzing the appearance and intensity of the 6K2-mCherry signal in neighboring cells without GFP-HDEL. In the wild-type Col-0, no cell-to-cell movement was observed at 7 dpi (Fig. 2, A and H), but a significant movement of 6K2-mCherry (30% of examined neighboring cells) was observed at 11 dpi (indicated by arrows in Fig. 2, C and H) and reached 90% at 15 dpi (Fig. 2, E and H), as judged by the percentage of neighboring cells showing 6K2-mCherry without GFP-HDEL. In rhd3-8, only ∼12% of neighboring cells had cell-to-cell movement of 6K2-mCherry at 11 dpi (Fig. 2, D and H). Although the number of cells expressing 6K2-mCherry increased at 15 dpi (Fig. 2, F and H), only 50% of neighboring cells without GFP-HDEL showed 6K2-mCherry. This result indicated that the cell-to-cell movement of the 6K2-mCherry vesicles is compromised in rhd3-8. Moreover, when the intensity of the intercellularly trafficked 6K2-mCherry vesicles was quantified, although the abundance of the 6K2-mCherry vesicles in the adjacent cells increased gradually in both the wild type and rhd3-8, the intensity of the 6K2-mCherry vesicles in rhd3-8 was always statistically significantly lower than that in the wild-type Col-0 (Fig. 2I). This result indicated that the production of 6K2-mCherry vesicles in secondary infected cells is also impaired.

The Formation and Maturation of Viral Vesicles Are Delayed in rhd3-8

We wanted to understand the subcellular basis of the affected TuMV replication and impaired cell-to-cell movement of the 6K2-tagged vesicles in rhd3-8. To this end, we used a confocal laser scanning fluorescent microscope to monitor the development or maturation of 6K2-GFP vesicles in Col-0 and rhd3-8 plant cells systemically infected with TuMV::6K2-GFP. As described in Jiang et al. (2015), in the wild-type Col-0 plant cells, the generation of small 6K2-GFP vesicles was observed at 9 dpi (Fig. 3A). At 11 dpi, larger ring-like vesicular structures (Fig. 3C, white arrows and inset) as well as the aggregation of small vesicles (Fig. 3C, red arrows) were seen. At 15 dpi, the aggregation of ring-like vesicular structures (Fig. 3E, white arrows and inset) and the aggregation of small vesicles (Fig. 3E, red arrows) became dominant and were clearly visible. In rhd3-8, fewer small viral vesicles were observed at 9 dpi than in the wild-type Col-0 (Fig. 3, B and G). Many fewer ring-like vesicular structures and less aggregation of small vesicles were observed at 11 and 15 dpi than at the same stage of the infected wild-type Col-0 (compare Fig. 3, D–C and F–E). We quantified the number of different 6K2-tagged vesicles based on their size (Fig. 3G) in both the wild-type Col-0 and rhd3-8. This process confirmed that although small vesicles with a size less than 2 μm were formed in both the wild type and rhd3-8 in the early stages of the infection, fewer such vesicles were seen in rhd3-8 (Fig. 3G). More importantly, compared to the ratio of various vesicles found in the wild-type Col-0 (Fig. 3G), we saw an increase in rhd3-8 in small vesicles at the expense of vesicles larger than 2 μm, or aggregation of small vesicles, at 9, 11, and 15 dpi (Fig. 3G). These results suggested that in rhd3-8, the formation as well as the development or maturation of 6K2-GFP vesicles into larger vesicular structures, or the aggregation of small vesicles during TuMV::6K2-GFP infection, is impaired in rhd3-8.

Figure 3.

Figure 3.

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The formation and maturation of viral vesicles are impaired in rhd3-8. A to F, Expression of TuMV:6K2-GFP during systemic infection in Col-0 (A, C, and E) and rhd3-8 (B, D, and F) plants at 9 dpi (A, B); 13 dpi (C, D); and 15 dpi (E, F). White arrows and insets show the ring-like structures, and red arrows show vesicles’ aggregation. Scale bars = 10 μm. Scale bars in sets = 2.5 μm. G, Number of 6K2-tagged vesicles per frame (n = 15 frames from four different plants of each treatment), in difference sizes as indicated in systemically infected cells of Col-0 and rhd3-8 plants at 9 dpi, 11 dpi, and 15 dpi. Each error bar represents ±se.

Transmission Electron Microscopy Confirmed that Maturation of Viral Vesicles Is Delayed in rhd3-8

To further confirm that there is impaired formation and development or maturation of TuMV-induced vesicles in rhd3-8, a transmission electron microscopy (TEM)-based time-course analysis was conducted of the production of various TuMV vesicles. According to Wan et al. (2015a), in the early stages of TuMV infection, convoluted membrane structures (CM) connected to rough ER are formed, a process that is followed by the production of SMVs at the mid-stage of infection. Both SMVs and DMVs with electron-dense cores are then found later in the infection process, followed by the formation of multivesicular bodies (MVBs) and vesicle aggregation (Wan et al., 2015a). As indicated in Figure 4, at 9 dpi in the wild-type Col-0, many SMVs—even DMVs—were observed, though some CMs still connected to the ER were also observed (Fig. 4, A and G). On the other hand, the majority of vesicles observed in rhd3-8 were CMs connected to the ER; although there were SMVs, fewer were produced (Fig. 4, B and G). At 11 dpi, the infection was advanced in Col-0, with a large increase in the abundance of individual SMVs and DMVs in the cytoplasm and less than 3.5% CMs (Fig. 4, C and G). In rhd3-8 cells, although the number of SMVs and DMVs were increased, ∼23% of vesicles are CMs still connected to the ER (Fig. 4, D and G). At 15 dpi, the progression of TuMV infection in the wild type continued with an even greater abundance of individual SMVs and DMVs, as well as the formation of MVBs (Fig. 4, E and G). In rhd3-8, the magnitude of increase of SMVs, especially DMVs and MVBs, was still less than that found in the wild-type Col-0, and still roughly 10% of vesicles were CMs (Fig. 4, F and G). These results indicated that the formation of TuMV viral vesicles (in particular, the development or maturation of TuMV CMs into SMVs, DMVs, and MVBs) are impaired in rhd3-8 mutants.

Figure 4.

Figure 4.

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TEM confirmed that maturation of viral vesicles is delayed in rhd3-8. A to F, TEM images of systemically TuMV-infected cells of Col-0 (A, C, and E) and rhd3-8 (B, D, and F) plants at 9 dpi (A, B); 11 dpi (C, D); and 15 dpi (E, F). Arrows show convoluted membrane structures (CM), single-membrane vesicles (SMV), double-membrane vesicles (DMV), and multivesicular bodies (MVB). Scale bars = 100 nm. G, Number of various TuMV replication vesicles per frame (n = 30 frames from three different plants of each treatment), as indicated in systemically TuMV-infected cells of Col-0 and rhd3-8 plants at 9 dpi, 11 dpi, and 15 dpi. Each error bar represents ±se.

6K2 Physically Interacts with RHD3

We wanted to understand how RHD3 is involved in the development or maturation of TuMV CMs into SMVs, DMVs, and MVBs. The TuMV transmembrane protein 6K2 is responsible for the generation of various replication vesicles. A yeast two-hybrid split-ubiquitin system (Y2H-SUS) assay showed that 6K2 interacted with RHD3 (Fig. 5, A and B, row 4). No interactions were found between PLV-Cub-RHD3 and free NubG (Fig. 5, A and B, row 1), or between PLV-Cub-6K2 and P3N-PIPO-NubG (Movahed et al., 2017; Fig. 5, A and B, row 2). Self-interaction within RHD3 was revealed, as described in Chen et al. (2011); illustrated in Fig. 5, A and B, row 3). Moreover, bimolecular fluorescence complementation (BiFC) confirmed that in tobacco (Nicotiana tabacum) epidermal cells, there was an interaction between YFPn-6K2 and YFPc-RHD3 (Fig. 5F). No interaction was observed when YFPn-6K2 was coexpressed with unfused YFPc (Fig. 5C); or when YFPc fused with P24α2d (Fig. 5D), an ER localized protein (Chen et al., 2011); or when YFPc-RHD3 was coexpressed with unfused YFPn (Fig. 5E).

Figure 5.

Figure 5.

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The 6K2 protein of TuMV physically interacts with RHD3. A and B, PLV-Cub-6K2 interacts with RHD3-NubG by yeast two-hybrid assay. Negative control: PLV-Cub-RHD3 and free NubG, PLV-Cub-6K2 and P3N-PIPO-NubG. Positive control: PLV-Cub-RHD3 and RHD3-NubG. The mated cells in (A) were grown on SC-Leu-Trp media. The mated cells in (B) were grown on SC-Leu-Trp-His containing (2 mM) 3AT. C, Coexpression of 6K2-CFP (red) with BiFC: YFPn-6K2 and YFPc (used as negative control) in tobacco. Scale bar, 10 μm. D, Coexpression of 6K2-CFP (red) with BiFC: YFPn-6K2 and YFPc-P24α1d (used as negative control) in tobacco. Scale bar = 10 μm. E, Coexpression of mCherry-RHD3 (red) with BIFC: YFPn and YFPc-RHD3 (used as a negative control) in tobacco. Scale bar = 10 μm. F, Coexpression of 6K2-CFP with BIFC: YFPn-6K2 and YFPc-RHD3 (green) in tobacco. Scale bar = 10 μm.

6K2 Is Able to Redirect RHD3 to 6K2 Vesicles

RHD3 is localized on the ER (Chen et al., 2011). Interestingly, during the TuMV infection, punctae of RHD3 were progressively observed along ER tubules during virus infection (Fig. 6, A–D). We found that the majority of RHD3 punctae colocalized with 6K2-GFP (Fig. 6, B–D). Along the development of the TuMV infection, the RHD3 punctae were colocalized with not only the punctate 6K2-GFP vesicles (Fig. 6, B–D, red arrows), but also the ring-like structures (Fig. 6, C and D, white arrows). The whole-image-based Pearson’s correlation coefficient (PCC) between RHD3 and 6K2 indicated that the colocalization between RHD3 and 6K2 increased along the development of the TuMV infection (Fig. 6E). We thus concluded that 6K2 can redirect RHD3 from the ER to viral replication vesicles.

Figure 6.

Figure 6.

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The 6K2 protein redirects RHD3 to 6K2 vesicles. A to D, Coexpression of TuMV::6K2-GFP with mCherry-RHD3 in tobacco plants at 7 dpi (A); 9 dpi (B); 11 dpi (C); and 13 dpi (D). White arrows and insets show ring-like structures, and red arrows show vesicles. Scale bar = 5μm. E, Quantification of colocalization between TuMV::6K2-GFP and mCherry-RHD3. The y axis is the PCC at 7 dpi, 9 dpi, 11 dpi, and 13 dpi; PCC was measured from the whole image (n = 20). Each error bar indicates ±se.

6K2GV, a Golgi-localized, Nonproductive 6K2 Mutant, Is Unable to Interact and Redirect RHD3 to 6K2 Vesicles

It has been observed that the predicted transmembrane domains of 6K2 contains a GxxxG motif (“x” being any amino acid) that is vital for TuMV infection and is needed to produce replication vesicles (Cabanillas et al., 2018). Replacement of the Gly residues with valines within the motif blocked the production of replication vesicles and delocalized 6K2 to Golgi bodies (Cabanillas et al., 2018). The generated mutant is designated as 6K2GV (Cabanillas et al., 2018). We found that unlike 6K2, 6K2GV did not interact with RHD3 in the BiFC assay (Fig. 7, A and B). The expression of 6K2GV-GFP generated punctate structures that were homogenous in size and almost all of them colocalized with the Golgi marker ST-mRFP (Cabanillas et al., 2018; Fig. 7C, blue arrows). No colocalization was found between 6K2GV and RHD3 (Fig. 7C, red arrows). These observations indicated that 6K2GV-GFP had lost its ability to form viral vesicles and became a default membrane protein transiting to the Golgi apparatus. It is unable to redirect RHD3 from the ER to the viral replication vesicles (Fig. 7C).

Figure 7.

Figure 7.

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A Golgi-localized 6K2 mutant, 6K2GV, is unable to interact with RHD3 and redirect it to 6K2 vesicles. A and B, Assessment of the interaction between the RHD3 and 6K2 (A) or 6K2GV (B) proteins via BiFC assay. A, coexpression of 6K2-mCherry (red) with BiFC: YFPc-6K2 and YFPn-RHD3 (green) in tobacco. B, coexpression of 6K2GV-mCherry (red) with BiFC: YFPc-6K2GV and YFPn-RHD3 (green) in tobacco. C, Coexpression of CFP-RHD3 (magenta) with 6K2GV-GFP (green) and ST-mRFP (yellow). Note the colocalization between 6K2GV-GFP and ST-mRFP (blue arrows), but no CFP-RHD3 is colocalized with 6K2GV-GFP and ST-mRFP (red arrows). D, Coexpression of CFP-RHD3 (magenta) with TuMV::6K2-GFP (green) and ST-mRFP (yellow) in tobacco plants. Note the colocalization between CFP-RHD3 and TuMV::6K2-GFP, but not with ST-mRFP (red arrows). Also note some colocalization between TuMV::6K2-GFP and ST-mRFP, but not CFP-RHD3 (blue arrows). Scale bars = 10 μm.

In TuMV infected cells, some 6K2 is known to be localized to the Golgi (Grangeon et al., 2012; Jiang et al., 2015). It is generally considered that those 6K2 transported to the Golgi are nonproductive for virus infection. By simultaneous visualization of CFP-RHD3 (magenta), TuMV::6K2-GFP (green), and Golgi stacks labeled by the trans-Golgi marker ST-mRFP (yellow), we found that the Golgi-localized 6K2 were not localized with RHD3 (Fig. 7D, blue arrows). Only non-Golgi localized 6K2-GFP in diverse sizes were colocalized with CFP-RHD3 punctae (Fig. 7D, red arrows).

DISCUSSION

As a positive-strand RNA virus, TuMV remodels the host ER for the formation of viral replication vesicles. Once these small viral vesicles are formed from the ER as CMs, they bud off from the ER exit sites and must bypass the Golgi to develop into their mature form for efficient viral replication and then move to the PD for cell-to-cell movement (Wan et al., 2015b). They may mature into replication competent SMVs (Wan et al., 2015a), or fuse with chloroplasts (Wei et al., 2013) for efficient replication, or mature into DMVs and MVBs for cell-to-cell movement in the infection process (Wan et al., 2015a). It has been shown that COPII protein SEC24A is required for the formation of TuMV replication vesicles (Hyodo et al., 2013; Wei et al., 2013; Jiang et al., 2015). SYP71, an ER localized SNARE protein is known to be involved in the fusion of TuMV-induced vesicles with chloroplasts (Wei et al., 2013).

In this study, we revealed that the formation of TuMV viral vesicles (in particular, the maturation of replication-incompetent viral-membrane structures formed in the ER into replication-competent SMVs, and later into DMVs and MVBs) is impaired in rhd3-8. Localized to ER membranes, RHD3 dimerizes to tether different ER membranes together, after which a conformational change of RHD3 dimers triggers an efficient homotypic fusion of different ER membranes (Sun and Zheng, 2018). The TuMV transmembrane protein 6K2, responsible for the formation and development of various viral vesicles, physically interacts with RHD3, possibly through the GxxxG motif in its transmembrane domain. The 6K2 protein can also redirect RHD3 from the ER to the various viral vesicle structures, including ring-like structures. We therefore think that during the infection process, RHD3 may be recruited (through the action of 6K2) to various viral vesicles for the maturation of CMs formed in the ER into SMVs, DMVs, and MVBs. We suspect that an efficient homotypic fusion of CMs budding off from the ER may be required for generation of SMVs, and that the homotypic fusion of SMVs is also necessary for the generation of DMVs.

It is known that there is tug-of-war between the formation and movement of various 6K2 viral vesicles and the transport of 6K2 to the Golgi (Cabanillas et al., 2018). It is generally considered that the transport of 6K2 to the Golgi is a nonproductive pathway for virus infection. It is interesting to note that in this study, those Golgi-localized 6K2 protein molecules are not localized with RHD3. Rather, only non-Golgi–localized 6K2-GFP punctae in diverse sizes are colocalized with the CFP-RHD3 punctae. Likely, RHD3 can be used as a marker for productive viral replication vesicles. Although our results indicated that RHD3 may play a role in the maturation of viral vesicles of TuMV, it is worth noting that the effect of RHD3 inactivation on TuMV systemic infection is relatively minor. We believe additional fusion factors are also involved in the formation of viral vesicles of TuMV. Cabanillas et al. (2018) showed that some ER-localized SNARE proteins do play a role in TuMV infection. It will be interesting to test if any of these SNAREs play a role in the maturation of TuMV vesicles like RHD3.

In rhd3-8, the replication of TuMV is impaired. In this regard, atlastin-1 was also reported to promote human immunodeficiency virus replication (Shen et al., 2017), but the underlying reason is known—it is known that SMVs are TuMV RNA replication sites (Wan et al., 2015a). Because the number and the percentage of SMVs are significantly lower in rhd3-8 plants than in the wild-type Col-0, we believe this reduction can account, at least in part, for the reduced replication of TuMV in rhd3-8. It will be interesting to see if altasin-1 also plays a similar role in human immunodeficiency virus replication.

In rhd3-8, not only the replication of TuMV is impaired; the intercellular movement of TuMV is also delayed. It has been reported that RHD3 is critical for intercellular trafficking of the Tomato spotted wilt tospovirus and its movement protein (Feng et al., 2016). In plant cells, the ER is an extended network of interconnected tubules and cisternae stretching throughout the cytoplasm. Because knockout of the RHD3 gene leads to the formation of a “cable-like” nonbranched ER network (Chen et al., 2011; Zhang et al., 2013; Feng et al., 2016), it was suggested that the affected ER structure plays a role in the delayed movement of the Tomato spotted wilt tospovirus and its movement protein (Feng et al., 2016). However, it is known that the replication vesicles of TuMV are not only the site of viral replication, but also the vehicle for TuMV intercellular movement (Movahed et al., 2017). It has been suggested that replication incompetent DMVs and MVBs may play an important role in the systemic spread of TuMV (Wan et al., 2015b). In the later stages of infection in infected cells, SMVs must mature into DMVs and MVBs for cell-to-cell movement. We found that such maturation is affected in rhd3-8 plants. We believe a delay in the evolution of SMVs to DMVs and MVBs may account, at least in part, for the delayed intercellular and systemic movement of TuMV.

MATERIALS AND METHODS

Plasmid Construction and Plant Materials

The construction of pCambiaTuMV::6K2-GFP and 6K2-CFP was described in Jiang et al. (2015). The dual TuMV::6K2-mCherry/GFP-HDEL construct was described in Grangeon et al. (2013). The construction of 6K2GV-mCherry and 6K2GV-GFP were described in Cabanillas et al. (2018). CFP-RHD3 was obtained by replacing GFP in pVKH18-GFP-RHD3 (Chen et al., 2011) with CFP. CFP in 6K2-CFP (Jiang et al., 2015) was amplified with the primers (X-CFP-FP: CCC​TCT​AGA​ATG​GTG​AGC​AAG​GGC​GAG​G and S-CFP-RP: CCCGTCGAC CTT​GTA​CAG​CTC​GTC​CAT​GCC). The PCR fragment and pVKH18-GFP-RHD3 were then digested with XbaI and SalI enzymes, purified and ligated with T4 DNA ligase. ST-mRFP in pVKH18En6 was provided by Federica Brandizzi (Michigan State University). The mCherry-RHD3 vector was created based on YFP-RHD3 in pEarleyGate104 with the recombination-based AQUA cloning method (Beyer et al., 2015). Both mCherry in 6K2GV-mCherry (Cabanillas et al., 2018) and the pEarleyGate 104 backbone were amplified with PCR with the primers (104-mCh-FP: ACA​AAC​AAC​ATT​ACA​ATT​ACA​TTT​A CAA​TTA​CCA​TGG​TGA​GCA​AGG​GCG​AGG​AG and 104-mCh-RP: TTCGAAGC TTG​AGC​TCG​AGA​TCT​GAG​TCC​GGA​CTT​GTA​CAG​CTC​GTC​CAT​GCC​G) and the primers (104-FP: GGT​AAT​TGT​AAA​TGT​AAT​TGT​AAT​GTT​GTT​TGT​TGT​TTG and 104-RP: TCC​GGA​CTC​AGA​TCT​CGA​GCT​C), respectively. The PCR products with the overlap hangers were then mixed and cotransformed together into DH5α cells. The mCherry-RHD3 positive colonies were then selected by colony PCR.

The construction of the PLV-Cub-6K2 plasmid for Y2H-SUS analysis was described in Jiang et al. (2015). The construction of PLV-Cub-RHD3 and NubG-RHD3 was described in Chen et al. (2011). The construction of P3N-PIPO was described in Movahed et al. (2017).

The construction of YFPn-6K2, YFPc-6K2, YFPc-P24α1d, and YFPc-RHD3 for BiFC analysis was described in Chen et al. (2011). The constructs were then transformed into Agrobacterium tumefaciens using a modified freeze/thaw procedure as described by Höfgen and Willmitzer (1988). The YFPc-6K2GV plasmid was provided by Daniel Garcia Cabanillas (INRS-Institut Armand-Frappier, Laval, Canada), and its construction was described in Cabanillas et al. (2018).

Six to eight-week-old plants of tobacco (Nicotiana tabacum) or Arabidopsis (Arabidopsis thaliana, Col-0 and rhd3-8) were used for all transient expression analyses and virus inoculations. Col-0 and rhd3-8 mutant seeds (SALK_025215) were obtained from the Arabidopsis Biological Resource Center (Ohio State University). Arabidopsis Col-0 and rhd3-8 seeds were selected on AT (Somerville and Ogren, 1982) growth medium. The seedlings on AT plates were planted in the soil after two weeks and grown at 20°C to 22°C under constant light. The tobacco seeds were grown directly on the soil at 20°C to 22°C under constant light.

Agrobacterium-Mediated Inoculation of Arabidopsis and Transient Expression in Tobacco

The TuMV::6K2-mCherry/GFP-HDEL, TuMV::6K2-GFP, 6K2-CFP, mCherry-RHD3, CFP-RHD3, 6K2GV-mCherry, 6K2GV-GFP, and ST-mRFP. plasmids were transformed into A. tumefaciens. The Agrobacterium containing the corresponding plasmids were grown in LB broth supplemented with kanamycin alone (for TuMV::6K2-mCherry/GFP-HDEL, TuMV::6K2-GFP, mCherry-RHD3, CFP-RHD3, and ST-mRFP) or kanamycin and ampicillin (for 6K2GV-mCherry, 6K2GV-GFP, and 6K2-CFP) overnight at 28°C with shaking. The cells were centrifuged at 2000 g for 10 min and resuspended in infiltration buffer (10 mm MgCl2 and 150 μm acetosyringone). The cell suspension was incubated for 4 h at room temperature before infiltration. The OD600 was adjusted to 0.40 for TuMV::6K2-GFP and TuMV::6K2-mCherry/GFP-HDEL, to 0.15 for 6K2-CFP and 0.02 for the rest. The agroinfiltration was done in 6 to 8-week-old Arabidopsis, the wild-type (Col-0), and rhd3-8 mutant, or tobacco.

Confocal Microscopy

Agroinfiltrated leaf sections were imaged using a Leica SP8 with the 63x immersion objective. Lasers of 448 nm, 488 nm, and 561 nm were used to excite CFP (YFP or GFP) and mCherry. The captures were done at 460 to 480 nm, 510 to 560 nm, 500 to 540 nm, and 580 to 620 nm, respectively. Image processing was done using Imaris image analysis software. To determine the number of 6K2-tagged vesicles of different sizes during systemic infection, stack images were taken from cells expressing TuMV::6K2-GFP. The image analysis was done using the Imaris software. All the frame sizes, sizes of the regions of interest, thresholds, surface areas, quality factors, and Z steps were the same for both Col-0 and rhd3-8 cells.

To study the production of 6K2-mCherry in primarily infected cells, we measured the mean intensity of 6K2-mCherry signals in cells expressing GFP-HDEL by using the MetaMorph microscopy automation and image analysis software from Molecular Devices. To quantify the intercellular movement of 6K2-mCherry in Col-0 and rhd3-8 plants after infiltration, we counted the percentage of cells with 6K2m-Cherry in cells neighboring GFP-HDEL cells and measured the mean intensity of 6K2-mCherry signals in red-only areas of the neighboring cells.

To assess the colocalization between TuMV::6K2-GFP and mCherry-RHD3, the PCC between the fluorescent signals was measured from whole images. PCC between sets of data are a measurement of how well the data are linearly related.

Yeast Two-Hybrid Split-Ubiquitin System Assay

The Y2H-SUS experiment was carried out as described by Grefen et al. (2007). The transformation of the yeast strains THY-AP4 and THY-AP5 (with respective plasmids used) was performed using a lithium-acetate based protocol (Grefen et al., 2007). Following transformation, the THY-AP4 and THY-AP5 strains containing the respective plasmids were mated and plated on SC-Leu-Trp (Grefen et al., 2007). Mated cells were then plated on the SC-Leu-Trp-His plates supplemented with 1 mm of the HIS3 competitive inhibitor 3-amino-1,2,4-triazole (3-AT; Brennan and Struhl, 1980).

Bimolecular Fluorescence Complementation (BiFC) Analysis

To study the interaction between the 6K2 and RHD3 proteins via BiFC analysis, different combinations of constructs, as indicated, were coexpressed by Agrobacterium-mediated transient expression at OD600 = 0.03 in tobacco leaf, the lower epidermal cells, as described by Sparkes et al. (2006). Fluorescence of YFP was then assessed 5 d postinfiltration using a Leica SP8 microscope with the 63x immersion objective. Lasers of 448 nm, 488 nm, and 561 nm were used to excite CFP, YFP, and mCherry, respectively. The emission captures were done at 460 to 480 nm, 510 to 560 nm, and 580 to 620 nm for CFP, YFP, and mCherry, respectively.

Transmission Electron Microscopy

Small pieces (1.5 mm x 2 mm) of TuMV systemically infected, upper noninfiltrated leaves of the wild-type Col-0 and rhd3-8 mutant plants were cut and fixed in 2.5% (w/v) glutaraldehyde in a 0.1 m sodium cacodylate buffer, pH 7.4, for 24 h at 4°C as described in Movahed et al. (2017). The sections were then examined in a Tecnai T12 transmission electron microscope (FEI) operating at 120 kV. Images were recorded using an AMT XR80C charge-coupled-device (CCD) camera system (FEI). Quantification of vesicles was done from a completely random collection of data from three different plants of each genotype at each time point, and with 30 totally randomly chosen frames with the same magnification.

Immunoblotting

To assess virus replication in primarily and systemically infected leaves, the leaves of 6-week-old wild-type Col-0 and rhd3-8 mutant plants were agroinfiltrated with the mock virus or pCambiaTuMV. At 12 d postinfection (dpi), 50 mg of the primarily and systemically infected leaves were ground in liquid nitrogen and mixed with 500 μl of SDS loading buffer. The proteins were extracted by boiling the mixture for 10 min at 100°C. After centrifugation for 1 min at 13,000 rpm, the supernatant was used for western blotting carried out with rabbit antisera anti-CP at 1:2500 dilution (Cotton et al., 2009) and anti-Rabbit IgG-peroxidase (Sigma-Aldrich) at 1:5000 dilution. The CP level was quantified using ImageJ (https://imagej.nih.gov/ij/). The total intensity of the bands was calculated by mean intensity of the bands multiplied by the area of each band. The ratio of the total intensity of each band in the test was divided by the total intensity of the bands derived from Coomassie blue staining.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: RHD3 (AT3G13870, 820600) and 6K2 of TuMV (1494058).

Acknowledgments

We would like to thank Federica Brandizzi (Michigan State University) for providing us with ST-mRFP in pVKH18En6. Our appreciation to Daniel Garcia Cabanillas (INRS, Laval, Canada) for providing us with 6K2GV-mCherry, 6K2GV-GFP, and YFPc-6K2GV. Also, we would like to express gratitude to the research group of the Facility for Electron Microscopy Research (FEMR) at McGill University in Montreal, Quebec, Canada, where we conducted the TEM experiments. We appreciate grant support from the Natural Sciences and Engineering Research Council of Canada and from the Quebec Fund for Research in Nature and Technology (to J.-F.L. and H.Z).

Footnotes

1

This work was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada and from the Quebec Fund for Research in Nature and Technology (to J.-F.L. and H.Z).

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