TY - JOUR
AU - Cheng, Chi-Ping
AB - Abstract NbRabF1, a small GTPase from Nicotiana benthamiana and a homolog of Arabidopsis thaliana Ara6, plays a key role in regulating Bamboo mosaic virus (BaMV) movement by vesicle transport between endosomal membranes. Reducing the expression of NbRabF1 in N. benthamiana by virus-induced gene silencing decreased the accumulation of BaMV, and with smaller infection foci on inoculated leaves, but had no effect in protoplasts. Furthermore, transient expression of NbRabF1 increased the accumulation of BaMV in inoculated leaves. Thus, NbRabF1 may be involved in the cell-to-cell movement of BaMV. The potential acyl modification sites at the second and third amino acid positions of NbRabF1 were crucial for membrane targeting and BaMV accumulation. The localization of mutant forms of NbRabF1 with the GDP-bound (donor site) and GTP-bound (acceptor site) suggested that NbRabF1 might regulate vesicle trafficking between the Golgi apparatus and plasma membrane. Furthermore, GTPase activity could also be involved in BaMV cell-to-cell movement. Overall, in this study, we identified a small GTPase, NbRabF1, from N. benthamiana that interacts with its activation protein NbRabGAP1 and regulates vesicle transport from the Golgi apparatus to the plasma membrane. We suggest that the BaMV movement complex might move from cell to cell through this vesicle trafficking route. BaMV, cell-to-cell movement, intracellular trafficking, Nicotiana benthamiana, Rab small GTPase Introduction Rab small GTPases (Rabs) play major roles in regulating communication between organelles via vesicular transport, which allows for exchanging lipids and membrane proteins (Goody et al., 2017; Langemeyer et al., 2018). Rabs cycle between the active (GTP-bound) and inactive (GDP-bound) forms, with GTP hydrolysis facilitated by GTPase-activating proteins (GAPs), and GDP replaced by GTP via the guanine nucleotide exchange factor (Pfeffer, 2013). The endocytic and vacuolar transport pathways in plant cells are different from non-plant cells, as some of the Rabs are unique (Ueda et al., 2001). Ara6 (RABF1) is a plant-specific Rab that is most similar to Ara7 (RABF2b), a conventional RabF2 ortholog in Arabidopsis (Rab5 in the animal system). Ara6 contains an extra stretch of amino acids at the N-terminus harboring the alkylating residues that target the membrane, whereas with conventional RabF2 proteins such as Ara7, the membrane targeting signal resides at the C-terminus. Both Ara6 and Ara7 localize on early endosomes, but Ara6 has also been observed on the plasma membrane (Ueda et al., 2001). Ara6 regulates vesicle trafficking between endosomes and the plasma membrane (Ebine et al., 2011). Rabs are involved in the infection process of animal pathogens including bacteria and viruses (Ireton et al., 2014; Mottola, 2014; Spano and Galan, 2018; Spearman, 2018), and different Rabs regulate various intracellular movements of animal viruses (Mannová and Forstova, 2003; Chambers and Takimoto, 2010; Mainou and Dermody, 2012; Hsiao et al., 2015; ). However, only a few cases have been reported in plant viruses. The movement protein of Cauliflower mosaic virus co-localizes with vesicles containing AtRAB-F2b, and is transported in an endocytic pathway (Carluccio et al., 2014). Rab5 is involved in recruiting the phosphatidylethanolamine-enriched membrane to build up the virus replication complex during Tobacco bushy stunt virus infection (Xu and Nagy, 2016). A Rab5 ortholog in Arabidopsis, Ara7, is involved in trafficking movement proteins of Potato mop-top virus in the early endocytic pathway (Haupt et al., 2005). Bamboo mosaic virus (BaMV) is a flexuous rod virus belonging to the Potexvirus genus of the Alphaflexiviridae family (Lin et al., 1977). The genome of BaMV is approximately 6.4 kb with a 5′ m7GpppG structure and a 3′ poly(A) tail; the viral RNA contains five open reading frames (ORFs) (Lin et al., 1994). ORF1 encodes a 155 kDa polypeptide for viral RNA replication with three functional domains: the capping enzyme domain containing the GTP methyltransferase and S-adenosylmethionine-dependent guanylyltransferase activities (Li et al., 2001a; Li et al., 2001b; Huang et al., 2004); the helicase-like domain containing the nucleoside triphosphatase and RNA 5′-triphosphatase activities (Li et al., 2001b); and the polymerase domain containing RNA-dependent RNA polymerase (RdRp) activity (Li et al., 1998; Li et al., 2001b). ORFs 2–4 encode three overlapping movement proteins (termed triple gene block protein, TGBp) for viral movement (Lin et al., 2004; Lin et al., 2006; Chen et al., 2012). ORF5 encodes a viral capsid protein required for viral RNA encapsidation, movement and symptom development (Lan et al., 2010; Lee et al., 2011; Hung et al., 2014a; Hung et al., 2014b). Previously, we identified a Rab GTPase activation protein 1 from Nicotiana benthamiana (NbRabGAP1) involved in BaMV cell-to-cell movement (Huang et al., 2013). The results suggested that NbRabGAP1 might regulate a Rab associated with vesicle trafficking, assisting BaMV movement. BaMV movement could involve shuttling between the plasma membrane and endoplasmic reticulum (ER) or early endosomes (Chen et al., 2017). Therefore, the possible candidate to interact with NbRabGAP1 would be a RabF1, a Rab5 ortholog. The main goal of this study was to identify which Rab is involved in regulating BaMV movement. Here, we identified one of the N. benthamiana RabF1 proteins involved in BaMV movement, and found that the membrane-targeting and GTPase activity of this RabF1 protein was critical for BaMV intercellular movement. Materials and methods Plants and viruses Nicotiana benthamiana plants were grown in a growth chamber with 16 h light/8 h dark at 28 °C, as previously described (Cheng et al., 2010). Virion or viral RNA of the BaMV severe strain (BaMV-S) (Lin et al., 1994) was used for inoculations. Constructs The TRV-knockdown system has been described elsewhere (Huang et al., 2016). In brief, the fragment from nt 379 to 594 of NbRabF1 was used for virus-induced gene silencing (VIGS). The fragment from nt 1 to 233 was used to determine the silencing efficiency. For transient expression, the cDNA of NbRabF1 was amplified by using the primers BamHI/NbRabF1/F (5′-GGGGGATCCATGGGTTGCGCATCTTCAGTTG-3′) and KpnI/NbRabF1/R (5′-GGGGGTACCAGCAGCAGACGGGCG-3′), and cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). The resulting clone was digested with BamH1 and KpnI, and cloned into pBin-mGFP with GFP fused at the C-terminus of NbRabF1. For the membrane-targeting mutants, the forward primers BamHI/NbRabF1G2A/F (5′-GGGGGATCCATGGCTTGCGCATCTTCAGTTG-3′), BamHI/NbRabF1C3A/F (5′-GGGGGATCCATGGGTGCCGCATCTTCAGTTG-3′) and BamHI/NbRabF1G2AC3A/F (5′-gggGGATCCATGGCTGCCGCATCTTCAGTTG-3′) containing the mutation sequences, and the reverse primer KpnI/NbRabF1/R were used to create G2A, C3A and G2AC3A mutants, respectively. To clone the GTP- and GDP-bound mutants, Q92L and S46N, the primers BamHI/NbRabF1/F and NbRabF1/Q92L/R (5′-GCGTACCTCTCCAGACCCG-3′) or NbRabF1/S46N/R (5′-GCAAAACAATACAGTTTTTACCAAC-3′), respectively, were used in PCR to generate mega-primers. The mega-primers were gel-eluted and used with KpnI/NbRabF1/R for second round PCR. VIGS and virus inoculation The TRV-based silencing system used for VIGS has been described previously (Huang et al., 2016). In short, the NbRabF1 fragment was inserted into a TRV2-containing vector and transformed into Agrobacteria C58C1. TRV1 (containing virus RdRp)- and TRV2-harboring Agrobacteria were mixed at a 1:1 ratio and infiltrated into the second to fourth true leaves of N. benthamiana at the five-leaf stage. Eighth and ninth true leaves were inoculated with 100 ng BaMV viral particles and harvested at 5 d post-inoculation (dpi). The inoculated leaves were harvested from each individual plant for further analysis, and at least three plants were analyzed for each treatment. Measurement of GFP foci As mentioned in the previous section, the leaves of knock-down plants were inoculated with 100 ng BaMV-GFP viral particles isolated from pCBG (an infectious viral vector carrying GFP gene and driven by 35S promoter)-inoculated leaves (Huang et al., 2016), and harvested at 5 dpi. The GFP foci on inoculated leaves were detected by fluorescent microscopy (Olympus IX71, Olympus, Japan) with excitation wavelength 488 nm. The size of each focus was measured by using the software Cellsens (Olympus, Japan). Protoplast isolation from knock-down plants and viral RNA inoculation The eighth and ninth true leaves from knock-down plants were harvested for protoplast isolation at 14 d post-Agrobacterium-infiltration. Approximately 1 μg BaMV viral RNA was inoculated into isolated protoplasts, as previously described (Huang et al., 2016). Total proteins were extracted at 24 h post-inoculation of protoplasts. Western blot analysis was used to determine the accumulation of BaMV coat protein (CP). Transient expression of NbRabF1 and its derivatives In general, Agrobacteria containing NbRabF1 or its derivatives were mixed with Agrobacteria harboring the silencing suppressor HcPro (Ivanov et al., 2016) at a 1:1 ratio and infiltrated onto the third to fifth leaves at the six-leaf stage. One day after infiltration, 100 ng BaMV virion was inoculated into infiltrated leaves and harvested at 5 dpi. The expression protocol has been described previously (Cheng et al., 2013). Fractionation of NbRabF1 and its derivatives NbRabF1 and its derivatives (NbRabF1/G2A, -/C3A or -/G2AC3A) were transiently expressed by Agrobacterium infiltration into N. benthamiana leaves at the seven-leaf stage. Approximately 0.5 g of infiltrated leaves was collected at 3 d post-infiltration. Total proteins were isolated by homogenizing the leaves (Retsch MM400 Mixer Mill,Thermo Fisher Scientific, MA, USA) with 1 ml pre-chilled buffer A (10 mM sodium phosphate, pH 7.4, 100 mM NaCl) containing 2 mM β-mercaptoethanol and 1 × protease inhibitor cocktail (Roche, Basel, Switzerland). The samples were centrifuged at 12 000×g for 5 min at 4 °C. The supernatant was used as the cytosol fraction. The pellet was washed twice with 1 ml buffer A and centrifuged at 12 000×g for 5 min at 4 °C to remove the washing solution. After washing, the pellet was stirred gently with 1 ml buffer A containing 2% Triton X-100 for 30 min at 4 °C, then centrifuged at 12 000×g for 5 min at 4 °C. The supernatant was collected as a membrane fraction. Western blot analysis was used to quantify the amount of each fraction of NbRabF1 and its derivatives (Cheng et al., 2013). Western blot analysis Total proteins were extracted from BaMV-inoculated leaves with extraction buffer (50 mM Tris-HCl pH 8.0, 10 mM KCl, 10 mM MgCl2, 1 mM EDTA, 20% glycerol, 2% SDS, 10% β-mercaptoethanol), and separated by 12% SDS PAGE. The BaMV CP was detected by western blot analysis using rabbit anti-BaMV CP polyclonal antibodies (generated in lab). Rabbit anti-GFP antibody was used to detect GFP-fused NbRabF1 and its derivatives. Rabbit anti-actin antibody (Yao-Hong Biotechnology Inc., Taiwan) was used to detect endogenous β-actin for normalization. Fluorescent-labeled anti-rabbit IgG (Rockland Immunochemicals Inc., Gilbertsville, PA, USA) was used as a secondary antibody. The fluorescent signal on the membrane was visualized and quantified by using LI-COR Odyssey (LI-COR Biosciences, Lincoln, NE, USA). The Rubisco large subunit (rbcL) was stained with Coomassie brilliant blue in the gel, for normalization in the transient expression experiment. Sub-cellular localization by laser scanning confocal microscopy Agrobacteria containing pBIN/AtARA6-OFP, -/NbRabF1-GFP, -/NbRabF1S46N-GFP, -/NbRabF1Q92L-GFP, -/NbTRXh2-OFP (plasma membrane marker) (Chen et al., 2017), pCD3-967 (Golgi mCherry marker), pCD3-975 (tonoplast marker) (Nelson et al., 2007), and pBIN61/HcPro were cultured and induced with 450 μM acetosyringone in 10 mM MgCl2 to obtain a final value of OD600=1. Agrobacteria harboring the pBIN/NbRabF1-GFP and its derivatives were mixed pairwise with the above organellar markers and that containing pBIN61-HcPro in a 1:1:1 ratio. All mixtures were infiltrated into N. benthamiana leaves, and 3 d post-infiltration, protoplasts were isolated from infiltrated leaves. Images were obtained by using an Olympus Fluoview FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan) with laser excitation wavelengths of 488 nm and 543 nm for GFP and mCherry, respectively, and 515 nm for YFP and OFP. Immunoprecipitation assay Agrobacteria containing pBIN/YFP, -/AtARA6-YFP, -/YFP-AtARA7, -/GFP, -/NbRabF1-GFP or -/NbRabF1/Q92L-GFP were mixed with Agrobacteria containing pBIN61/HcPro and -/NbRabGAP1-T7 in a 1:1:1 ratio, respectively, and infiltrated into N. benthamiana leaves. At 3 d post-infiltration, the GFP/YFP-containing proteins from transiently expressed leaves were extracted with buffer containing 20 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 300 mM NaCl, 5 mM DTT, 2.5% PVPP and 1 × protease inhibitor (Roche), and purified by using the GFP-Trap agarose kit (Chromotek, Martinsried, Germany). The pull-down proteins were analyzed by western blot analysis. Yeast two-hybrid assay The gene fragment of NbRabGAP1 or the interacting domain (TBC) NbRabGAP1/TBC was cloned into the prey plasmid pYESTrp, and NbRabF1 or GTP-bound mutant NbRabF1/Q92L was cloned into the bait plasmid pHyLex/Zeo (Invitrogen, Carlsbad, CA, USA). The proteins of NbRabGAP1 or NbRabGAP1/TBC and NbRabF1 or NbRabF1/Q92L were co-expressed in Saccharomyces cerevisiae strain L40, and selected on Trp-/Zeocin plates. The interaction was tested on Trp-/His-/Zeocin plates when pYESTrp-Jun and pHyLEX/Zeo-Fos were used as positive controls, and pYESTrp-Jun and pHyLEX/Zeo-Lamin as negative controls. Expression and purification of His-tagged proteins from E. coli The coding sequence of NbRabF1 was amplified with the forward primer KpnI-5′+1 RabF1 (5′-GGTACCATGGGTTGCGCATCTTCAGT-3′) and the reverse primer XhoI-3′-RabF1 (5′-GCTCGAGAGCAGCAGACGGGCGTGGTAA-3′) (KpnI and XhoI sites). The PCR product was cloned into the pGEM-T easy vector (Promega) and verified by sequencing. Finally, NbRabF1 was sub-cloned from the T-vector into the pET29a (+) expression vector (Invitrogen) and transformed into E. coli BL21(DE3). The resulting clone was designated pET29a (+)-NbRabF1. E. coli containing pET29a(+)-NbRabF1 was cultured to OD600=1 (120 ml in total volume), the expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside at 16 °C for 24 h, then samples were centrifuged at 5927 ×g at 4 °C for 7 min. The cell pellet was resuspended in 8 ml buffer A (50 mM Tris-HCl pH 7.5, 150 mM NaCl and 2 mM DTT) containing protease inhibitor cocktail (Roche) and subjected to sonication (Sonicator 3000, Misonix, NY, USA) at 4 °C with 15% amplitude at 10 s intervals for 20 min. The cell extract was clarified by centrifugation at 17 418 ×g for 5 min, then incubated with 1 ml High-Capacity Profinity™ IMAC Resins (Bio-Rad, CA, USA) at 4 °C for 1 h, followed by washing with 10 ml buffer A containing 25 mM imidazole, and eluted with buffer A containing 250 mM imidazole. Finally, the eluted protein was passed through Sephacryl S-300 gel filtration system at a flow rate of 1 ml min-1 and dialyzed with buffer containing 20 mM Tris-HCl pH 8.0, 50 mM NaCl, and 10% glycerol. The control His-GFP construct was manipulated under the same conditions. GTPase activity The GTPase activity of NbRabF1 was analyzed with Transcreener® GDP FI assay (BellBrookLabs, Madison, WI, USA) as previously described (Hoepflinger et al., 2013). The purified recombinant protein of NbRabF1 GTPase was applied in the assay. When the reaction product GDP is released and displaced by Alexa Fluor 594-GDP tracer bound to a GDP antibody–IR dye quencher conjugate, the signal of Alexa Fluor 594 can be detected and quantified by SpectraMax M2 (Molecular Devices, CA, USA). The excitation wavelength is 584 nm and the emission wavelength is 610 nm. The recombinant proteins were diluted with the buffer containing 50 mM HEPES (pH 7.5), 4 mM MgCl2, 2 mM EGTA, 1% DMSO, and 0.01% Triton X-100. Approximately 1.5 μM recombinant protein was applied to the assay with or without 10 μM GTP. All measurements were performed in triplicate at around 28 °C according to manufacturer’s instructions. BiFC assay To construct NbRabF1 for the BiFC assay, GFP and OFP sequences on pBIN-NbRabGAP1-OFP, pBIN-NbRabF1-GFP and pBIN-NbRabF1Q92L-GFP were replaced with nYFP and cYFP sequences, respectively. The primer set for cloning nYFP and cYFP was KpnI-nYFP (5′-GGTACCATGGTGAGCAA-3′)/ SacI-nYFP (5′-GAGCTCTCAGTCCTCGATGT-3′), and KpnI-cYFP (5′-GGTACCATGGGCAGCGTGCA-3′)/SacI-cYFP (5′-GAGCT CTCACTTGTACAGCT-3′), respectively. Agrobacteria containing pBIN-NbRabGAP1-nYFP was mixed with that containing pBIN-NbRabF1-cYFP or pBIN-NbRabF1Q92L-cYFP in a 1:1 ratio and infiltrated into N. benthamiana leaves for 3 d, while the pBIN-nYFP/-cYFP, pBIN-NbRabGAP1-nYFP/-cYFP, pBIN-nYFP/- NbRabf1-cYFP, and pBIN-nYFP/-NbRabf1Q92L-cYFP were used as negative controls. On the other hand, Agrobacteria containing pBIN-NbRabF1-nYFP or pBIN-NbRabF1Q92L-nYFP was mixed with that containing pBIN-NbRabGAP1-cYFP in a 1:1 ratio to confirm the interaction between NbRabGAP1 and NbRabF1. Images were obtained with an Olympus Fluoview 3000 laser scanning confocal microscope using a laser excitation of 514 nm for YFP. Results Identification of two RabF1 isoforms in N. benthamiana Results from previous studies indicated that BaMV movement was regulated by a Rab-GTPase–activating protein NbRabGAP1 (Huang et al., 2013), and might be trafficked from the ER membrane to plasma membrane (Chen et al., 2017). The possible Rab candidate involved in vesicle trafficking in this route was identified as RABF (Ueda et al., 2001; Ebine et al., 2011). To investigate whether a member of the RABF family is the target of NbRabGAP1 from N. benthamiana that could be involved in BaMV infection, the plant-specific RabF1 sequence from Arabidopsis AtAra6 (accession number: AB007766) (Altschul et al., 1997) was used to search the N. benthamiana database (http://benthgenome.qut.edu.au/). Two sequences, NbRab6a and NbRab6b, with only one amino acid difference were obtained (Fernandez-Pozo et al., 2015), and aligned with that of AtAra6 (Fig. 1A). On sequence alignment, NbRab6a and AtAra6 showed 87% identity. We cloned the coding region of the two genes, NbRab6a and NbRab6b, and expressed it in N. benthamiana leaf cells. Using sub-cellular localization analysis, NbRab6a-GFP and NbRab6b-OFP were found to co-localize (Fig. 1B). Thus, the two isoforms could be functionally identical. The sequence of NbRab6a was then used to represent NbRabF1 for further analysis. Fig. 1. Open in new tabDownload slide Amino acid sequence alignment and sub-cellular localization of NbRab6. (A) The sequence alignment of Nicotiana benthamiana NbRab6a (Niben101Scf29276g00003.1 from Sol Genomics Network) and NbRab6b (Niben101Scf00648g00003.1 from Sol Genomics Network) and Arabidopsis thaliana AtAra6 (GenBank: AB007766). (B) Sub-cellular localization of GFP-fused NbRab6a and OFP-fused NbRab6b co-expressed in N. benthamiana leaves. Protoplasts were isolated from leaves for confocal imaging. GFP is in green, and OFP is in red. Scale bar=10 μm. Fig. 1. Open in new tabDownload slide Amino acid sequence alignment and sub-cellular localization of NbRab6. (A) The sequence alignment of Nicotiana benthamiana NbRab6a (Niben101Scf29276g00003.1 from Sol Genomics Network) and NbRab6b (Niben101Scf00648g00003.1 from Sol Genomics Network) and Arabidopsis thaliana AtAra6 (GenBank: AB007766). (B) Sub-cellular localization of GFP-fused NbRab6a and OFP-fused NbRab6b co-expressed in N. benthamiana leaves. Protoplasts were isolated from leaves for confocal imaging. GFP is in green, and OFP is in red. Scale bar=10 μm. NbRabF1 assists BaMV infection To investigate the effect of NbRabF1 on BaMV infection, we used Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to knock down the expression of NbRabF1. Because the region chosen for knocking down the expression of NbRabF1 is the same sequence as Rab6a and Rab6b, both genes, if expressed, would be knocked down with VIGS. The phenotype of NbRabF1-knock-down plants did not differ from that of control Luc-knock-down plants (Supplementary Fig. S1 at JXB online). The expression of NbRabF1 was reduced to 18% of that of the control Luc-knock-down leaves (Fig. 2A). The accumulation of BaMV coat protein (CP) in NbRabF1-knock-down leaves was reduced to 77% that of control leaves (Fig. 2B), with no effect on BaMV accumulation in NbRabF1-knock-down protoplasts (Fig. 2C). Thus, NbRabF1 is likely to be involved in BaMV cell-to-cell movement. To validate this hypothesis, we used fluorescent microscopy to measure the size of BaMV infection foci on NbRabF1-knock-down leaves. Because the infectious BaMV cDNA clone was constructed to express GFP, the infection foci represented by GFP fluorescence could be directly measured (Fig. 3A). The mean size of BaMV infection foci was smaller on leaves of NbRabF1-knock-down plants compared with Luc-knock-down plants, such that the leasion size was reduced to approximately 67% (Fig. 3B). Fig. 2. Open in new tabDownload slide Relative accumulation of BaMV coat protein (CP) in NbRabF1-knock-down plants and protoplasts. (A) Real-time qRT-PCR showing expression of NbRabF1 in Luc- and NbRabF1-knock-down leaves. The expression of NbRabF1 in control plants (Luc) was set to 100%. (B) and (C) Western blot analysis of BaMV CP accumulation on the inoculated leaves at 5 d post-inoculation (B), and in protoplasts at 24 h post-inoculation (C). The accumulation of BaMV CP in control plants or protoplasts was set to 100%. Data are the mean ±SD of relative concentrations of CP from the number of independent experiments indicated as N, with the number of plants indicated as n in each experiment. The panels below show representative western blot results of CP concentrations with Rubisco large subunit (rbcL) as a control (the loading control for normalization). ***P<0.001 by Student’s t-test. Fig. 2. Open in new tabDownload slide Relative accumulation of BaMV coat protein (CP) in NbRabF1-knock-down plants and protoplasts. (A) Real-time qRT-PCR showing expression of NbRabF1 in Luc- and NbRabF1-knock-down leaves. The expression of NbRabF1 in control plants (Luc) was set to 100%. (B) and (C) Western blot analysis of BaMV CP accumulation on the inoculated leaves at 5 d post-inoculation (B), and in protoplasts at 24 h post-inoculation (C). The accumulation of BaMV CP in control plants or protoplasts was set to 100%. Data are the mean ±SD of relative concentrations of CP from the number of independent experiments indicated as N, with the number of plants indicated as n in each experiment. The panels below show representative western blot results of CP concentrations with Rubisco large subunit (rbcL) as a control (the loading control for normalization). ***P<0.001 by Student’s t-test. Fig. 3. Open in new tabDownload slide Cell-to-cell movement of BaMV in Luc- and NbRabF1-knock-down leaves. (A) The area of fluorescent foci in inoculated leaves of Luc-knock-down control (Luc) and NbRabF1-knock-down (NbRabF1) plants after inoculation with the BaMV infectious plasmid expressing GFP. Scale bar=1 mm. Two foci are shown (upper and lower panels) for each treatment. (B) The quantification results were derived from (A). Data are mean ±SD of 86 and 72 foci from Luc- and NbRabF1-knock-down plants, respectively. *** P<0.001 by Student t-test. (This figure is available in color at JXB online.) Fig. 3. Open in new tabDownload slide Cell-to-cell movement of BaMV in Luc- and NbRabF1-knock-down leaves. (A) The area of fluorescent foci in inoculated leaves of Luc-knock-down control (Luc) and NbRabF1-knock-down (NbRabF1) plants after inoculation with the BaMV infectious plasmid expressing GFP. Scale bar=1 mm. Two foci are shown (upper and lower panels) for each treatment. (B) The quantification results were derived from (A). Data are mean ±SD of 86 and 72 foci from Luc- and NbRabF1-knock-down plants, respectively. *** P<0.001 by Student t-test. (This figure is available in color at JXB online.) Upon transient expression of NbRabF1 in N. benthamiana leaves (Fig. 4A) followed by BaMV inoculation, BaMV accumulation was increased to 137% that of the controls at 5 dpi (Fig. 4B). Together, the results from loss-of-function (knock-down experiments; Fig. 3) and gain-of-function (transient expression experiments; Fig. 4) indicated that NbRabF1 facilitated BaMV intercellular movement. Fig. 4. Open in new tabDownload slide The accumulation of BaMV in N. benthamiana leaves expressing NbRabF1 or its derivatives. (A) The protein expression of NbRabF1-OFP and its derivatives in N. benthamiana leaves was determined by western blot analysis using an antibody against OFP. (B) The relative accumulation of BaMV CP was determined by western blot analysis. Total protein was extracted from BaMV-inoculated leaves transiently expressed with OFP, NbRabF1-OFP, NbRabF1/S46N-OFP, NbRabF1/Q92L or NbRabF1/G2AC3A-OFP at 5 d post-inoculation. Data are the mean ±SE from three independent experiments. The expression of actin was used as a loading control. * P<0.05 by Student’s t-test. Fig. 4. Open in new tabDownload slide The accumulation of BaMV in N. benthamiana leaves expressing NbRabF1 or its derivatives. (A) The protein expression of NbRabF1-OFP and its derivatives in N. benthamiana leaves was determined by western blot analysis using an antibody against OFP. (B) The relative accumulation of BaMV CP was determined by western blot analysis. Total protein was extracted from BaMV-inoculated leaves transiently expressed with OFP, NbRabF1-OFP, NbRabF1/S46N-OFP, NbRabF1/Q92L or NbRabF1/G2AC3A-OFP at 5 d post-inoculation. Data are the mean ±SE from three independent experiments. The expression of actin was used as a loading control. * P<0.05 by Student’s t-test. Membrane targeting of NbRabF1 is required for BaMV accumulation Sequence analysis and comparison of NbRabF1 with other RABF1 members indicated that glycine and cysteine at the second amino acid (G2) and third amino acid (C3) positions, respectively, are conserved (Fig. 1A). The G2 and C3 residues were found to be N-myristoylated and palmitoylated, respectively, for membrane targeting (Ueda et al., 2001). To demonstrate that the membrane targeting of NbRabF1 is crucial for BaMV accumulation, we substituted the amino acids G2 and C3 for alanine to produce NbRabF1/G2A, -/C3A and -/G2AC3A. Fractionation analysis indicated that NbRabF1 was mainly localized in the membrane fraction (Fig. 5A). By contrast, most of NbRabF1/G2A was localized in the cytosol. However, NbRabF1/C3A remained equally distributed in the cytosol and membrane fractions (Fig. 5A). Thus, G2 myristoylation may be more critical than C3 palmitoylation for NbRabF1 to target the membrane. The double mutant NbRabF1/G2AC3A mostly distributed to the cytosol in the fractionation assay. Confocal data also confirmed these results: G2A was distributed in the cytoplasm and most of C3A remained in the membrane, whereas G2AC3A was localized to both cytoplasm and nucleus (Fig. 5B). The results suggested that G2-myristoylation and C3-palmitoylation of NbRabF1 are required for membrane targeting. Fig. 5. Open in new tabDownload slide Fractionation and sub-cellular localization of NbRabF1 and its targeting mutants. (A) Western blot analysis of NbRabF1-GFP and its targeting mutants -/G2A-GFP, -/C3A-GFP and -/G2AC3A-GFP in the cytoplasm (C) or membrane (M) fraction. (B) GFP only and NbRabF1-GFP and its derivatives were transiently expressed in N. benthamiana leaves. Protoplasts were isolated from these leaves and examined by confocal microscopy. GFP is shown in green and the autofluorescence of chloroplasts is in red. Scale bar=20 μm. Fig. 5. Open in new tabDownload slide Fractionation and sub-cellular localization of NbRabF1 and its targeting mutants. (A) Western blot analysis of NbRabF1-GFP and its targeting mutants -/G2A-GFP, -/C3A-GFP and -/G2AC3A-GFP in the cytoplasm (C) or membrane (M) fraction. (B) GFP only and NbRabF1-GFP and its derivatives were transiently expressed in N. benthamiana leaves. Protoplasts were isolated from these leaves and examined by confocal microscopy. GFP is shown in green and the autofluorescence of chloroplasts is in red. Scale bar=20 μm. The expression of NbRabF1/G2AC3A-GFP showed reduced ability to assist BaMV accumulation compared with the wild-type protein (Fig. 4). Thus, membrane targeting of NbRabF1 is critical for BaMV accumulation. The results also implied that the BaMV movement complex could be trafficked through vesicles with the assistance of NbRabF1. NbRabF1 regulates vesicle trafficking from the Golgi apparatus to plasma membrane To reveal the intracellular vesicle trafficking pathway of NbRabF1, we used a few known organellar markers that co-localize with NbRabF1. NbRabF1-GFP co-localized with Golgi (Man49)-mCherry and the endosomal marker AtAra6-OFP (Ueda et al., 2001; Haas et al., 2007) (Fig. 6). Based on the structural analysis of Rab proteins (Dumas et al., 1999; Lee et al., 2009; Lamers et al., 2017), the β1/α1 loop provides phosphate contacts and a Ser/Thr residue (corresponding to the S46 of NbRabF1) to coordinate Mg2+. Mutant S46N could disrupt the binding of Mg2+ essential for GTP binding and consequently lock the GTPase in an inactive form (GDP-bound). By contrast, mutant Q92L could affect the conserved residue critical for catalysis and consequently fix the GTPase in a GTP-bound form (Lamers et al., 2017). Therefore, to determine the donor and acceptor compartments of the vesicle associated with NbRabF1, based on structural analysis, we constructed mutant NbRabF1/S46N (GDP-bound form) and NbRabF1/Q92L (GTP-bound form) fused with GFP. NbRabF1/S46N representing the donor compartment co-localized with the Golgi marker; and NbRabF1/Q92L representing the acceptor compartment co-localized with the plasma membrane marker, NbTRXh2-OFP (Chen et al., 2018) (Fig. 6). From the confocal images, we propose that NbRabF1 could likely shuttle between the Golgi apparatus and plasma membrane through the endosomal membrane system. Fig. 6. Open in new tabDownload slide Sub-cellular localization of NbRabF1 and its derivatives with various organellar markers. The expression of GFP-fused NbRabF1 and its derivatives is in green. The organellar markers fused with OFP or mCherry are in red. Scale bar=20 μm. Fig. 6. Open in new tabDownload slide Sub-cellular localization of NbRabF1 and its derivatives with various organellar markers. The expression of GFP-fused NbRabF1 and its derivatives is in green. The organellar markers fused with OFP or mCherry are in red. Scale bar=20 μm. GTPase activity of NbRabF1 might be critical for efficient BaMV infection To examine whether the NbRabF1 we cloned harbors GTPase activity, we overexpressed and purified the protein from E. coli. The GTPase activity was then tested with the labeled-GDP fluorescence intensity readout assay (Hoepflinger et al., 2013). The results indicated that NbRabF1 has a relative GTPase activity higher than that of the control (the GFP-only construct expressed and purified from E. coli) (Supplementary Fig. S2). We then expressed NbRabF1/S46N (GDP-bound form at Golgi) and NbRabF1/Q92L (GTP-bound form at plasma membrane) in leaves, followed by BaMV inoculation. The expression of NbRabF1/S46N had no effect on BaMV accumulation compared to that of NbRabF1. By contrast, the expression of NbRabF1/Q92L had a negative effect on BaMV accumulation (Fig. 4B). These results suggest that the GTPase activity of NbRabF1 might be playing a critical role in assisting BaMV movement. Mutant NbRabF1/Q92L with the stabilized GTP-bound form, and the GTP failing to be hydrolyzed, could have a negative effect on BaMV accumulation. NbRabF1 interacts with NbRabGAP1 To examine whether NbRabGAP1 targets NbRabF1, we co-expressed NbRabGAP1-OFP and NbRabF1/Q92L-GFP in N. benthamiana protoplasts. The two proteins co-localized on the plasma membrane (Fig. 7A). To validate the interaction between NbRabGAP1 and NbRabF1, we used a co-immunoprecipitation assay (Fig. 7B) and yeast two-hybrid experiments (Fig. 7C). NbRabGAP1-T7 could be detected only when co-expressed with NbRabF1-GFP or NbRabF1/Q92L-GFP, but not GFP alone in N. benthamiana cells (Fig. 7B). The results of yeast two-hybrid experiments confirmed the interaction of NbRabGAP1 with NbRabF1 or NbRabF1/Q92L. The interaction through the TBC domain of NbRabGAP1 was also confirmed (Fig. 7C). Hence, BaMV may move in N. benthamiana cells via vesicle trafficking regulated by NbRabF1, activated by NbRabGAP1 on the plasma membrane. Fig. 7. Open in new tabDownload slide Sub-cellular localization and the interaction of NbRabGAP1 with NbRabF1. (A) OFP-fused NbRabGAP1 was co-expressed with NbRabF1/Q92L in N. benthamiana leaves. Protoplasts were isolated from these treated leaves for confocal imaging. GFP is in green, and OFP is in red. Scale bar=20 μm. (B) Total proteins (input) were extracted from N. benthamiana leaves and immunoblotted with antibodies against T7-tag for NbRabGAP1, and GFP for NbRabF1 and NbRabF1/Q92L, as indicated. Total proteins were then immunoprecipitated with anti-GFP beads (IP/GFP) and immunoblotted (WB) with the antibody against GFP or T7-tag (bottom). (C) Interaction of NbRabF1 with NbRabGAP1 in yeast cells. Yeast strain L40 co-transformed with the indicated plasmids was subjected to 10–fold serial dilution and incubated with minimal medium lacking tryptophan and histidine supplemented with Zeocin to identify protein interactions. Yeast containing pYESTrp-Jun and pHyLEX/Zeo-Fos was used as a positive control; yeast containing the vector pYESTrp-Jun and pHyLEX/Zeo-Lamin was a negative control. Fig. 7. Open in new tabDownload slide Sub-cellular localization and the interaction of NbRabGAP1 with NbRabF1. (A) OFP-fused NbRabGAP1 was co-expressed with NbRabF1/Q92L in N. benthamiana leaves. Protoplasts were isolated from these treated leaves for confocal imaging. GFP is in green, and OFP is in red. Scale bar=20 μm. (B) Total proteins (input) were extracted from N. benthamiana leaves and immunoblotted with antibodies against T7-tag for NbRabGAP1, and GFP for NbRabF1 and NbRabF1/Q92L, as indicated. Total proteins were then immunoprecipitated with anti-GFP beads (IP/GFP) and immunoblotted (WB) with the antibody against GFP or T7-tag (bottom). (C) Interaction of NbRabF1 with NbRabGAP1 in yeast cells. Yeast strain L40 co-transformed with the indicated plasmids was subjected to 10–fold serial dilution and incubated with minimal medium lacking tryptophan and histidine supplemented with Zeocin to identify protein interactions. Yeast containing pYESTrp-Jun and pHyLEX/Zeo-Fos was used as a positive control; yeast containing the vector pYESTrp-Jun and pHyLEX/Zeo-Lamin was a negative control. Furthermore, we also used bimolecular fluorescence complementation (BiFC) assay to visualize the interaction in live cells. The fluorescent signal could only be detected when NbRabGAP1-nYFP was co-expressed with NbRabF1-cYFP or NbRabF1/Q92L-cYFP, or reverse constructs (Fig. 8). Fig. 8. Open in new tabDownload slide Bimolecular fluorescence complementation (BiFC) assay. The amino-terminal fragment (nYFP) or carboxyl-terminal fragment (cYFP) of YFP was fused to the C-terminus of NbRabF1 (shown as RabF1-nYFP or RabF1-cYFP, respectively), NbRabF1/Q92L (shown as RabF1/Q92L-nYFP or RabF1/Q92L-cYFP, respectively), or NbRabGAP1 (shown as RabGAP1-nYFP or RabGAP1-cYFP, respectively). The paired co-expression of those fused with nYFP or cYFP was indicated above each panel. Images were obtained with an Olympus Fluoview 3000 laser scanning confocal microscope using 514 nm laser excitation for YFP. The DIC images are also shown under each fluorescent image. Fig. 8. Open in new tabDownload slide Bimolecular fluorescence complementation (BiFC) assay. The amino-terminal fragment (nYFP) or carboxyl-terminal fragment (cYFP) of YFP was fused to the C-terminus of NbRabF1 (shown as RabF1-nYFP or RabF1-cYFP, respectively), NbRabF1/Q92L (shown as RabF1/Q92L-nYFP or RabF1/Q92L-cYFP, respectively), or NbRabGAP1 (shown as RabGAP1-nYFP or RabGAP1-cYFP, respectively). The paired co-expression of those fused with nYFP or cYFP was indicated above each panel. Images were obtained with an Olympus Fluoview 3000 laser scanning confocal microscope using 514 nm laser excitation for YFP. The DIC images are also shown under each fluorescent image. Discussion Rabs are a group of small GTPases that regulate the formation of vesicles, through which materials such as cargo proteins and viruses can be transported between organelles. In this study, we identified a Rab, NbRabF1, involved in BaMV movement. The sub-cellular localization of NbRabF1 is similar to that of CaAra6 from green algae, which localizes to the Golgi apparatus, endosomes and plasma membrane (Hoepflinger et al., 2013). However, unlike AtAra6 (Ueda et al., 2001), NbRabF1 also co-localized with the Golgi (TGN) marker, which was further identified as the location of the GDP-bound form NbRabF1/S46N, indicated as a donor site of vesicle formation. The GTP-bound form NbRabF1/Q92L co-localized with the plasma membrane marker, indicated as the recipient site of vesicle transport (Fig. 5). These results suggest that NbRabF1-containing vesicles bud out from the Golgi apparatus and travel to the plasma membrane through the endosomal pathway. The BaMV viral movement complex might take advantage of this novel mechanism to traffic from the Golgi apparatus to the plasma membrane and then move towards plasmodesmata to move into another cell. Expression of the GDP-bound form NbRabF1/S46N had no effect on BaMV accumulation (Fig. 4). By contrast, the other mutant with a GTP-bound form, NbRabF1/Q92L, had a dominant negative effect when expressed. These results suggest that the NbRabF1/Q92L with a GTP-bound form might interact with the GAP protein and be docked on the plasma membrane. This situation could have an effect on recycling the endogenous NbRabF1 required for BaMV movement. However, this dominant negative effect was not observed on the other side of the trafficking pathway: NbRabF1/S46N with the GDP-bound form and docked on the donor membrane (Golgi), had no effect on BaMV trafficking (Fig. 4). It is possible that the endogenous NbRabF1 may be mostly on endosomal and Golgi membranes (Fig. 5) and recharging of the inactive form (GDP bound) to the active form (GTP bound) of NbRabF1 is not a critical step in transporting the cargos (including BaMV) to the plasma membrane. Therefore, the effect of retarding transportation was not observed when NbRabF1/S46N was expressed and trapped at the Golgi membrane (Fig. 4). The assembly of the BaMV movement complex is believed to be initiated at the ER membrane, where the two transmembrane viral movement proteins, TGBp2 and TGBp3 (Chou et al., 2013) are translated. The two movement proteins are recruited to assemble the viral movement complex containing viral RNA, TGBp1, replicase, and CP, at the ER or Golgi membrane. The viral movement complex then moves to the plasma membrane via the endosomal system by vesicle trafficking with the activation of NbRabF1. Although the details of viral movement complex trafficking to the plasma membrane is not clear, the host factor elicitor-inducible leucine-rich repeat receptor-like protein (NbEILP) is involved (Chen et al., 2017). NbEILP could carry a signal to be recruited to the NbRabF1-induced vesicle, and one of the components in the viral movement complex could interact with NbEILP. Therefore, the BaMV movement complex could hitch-hike the trafficking vesicle from the Golgi apparatus to the plasma membrane and then reach plasmodesmata. However, the viral complex on the plasma membrane could be negatively regulated by two host factors, the plasma membrane-associated cation-binding protein 1-like protein (Huang et al., 2017) and a thioredoxin, NbTRXh2 (Chen et al., 2018). Through the interaction of these two host proteins with the replicase in the viral movement complex, the cell-to-cell movement is retarded. This study aimed to identify a Rab together with its partner, NbRabGAP1 (Huang et al., 2013), in assisting BaMV cell-to-cell movement. In the co-immunoprecipitation assay, we revealed the physical interaction of the Ara6 homolog, NbRabF1, from N. benthamiana with NbRabGAP1 (Fig. 7). Two Rabs, NbRabG3f (Huang et al., 2016) and NbRabF1 (this study), and one GAP, NbRabGAP1 (Huang et al., 2013), participate in BaMV infection. These results suggested that the two Rabs localize on the Golgi apparatus and regulate vesicle trafficking. NbRabG3f mainly participates in BaMV replication and possibly its movement as well (Huang et al., 2013), and NbRabF1 is involved in BaMV movement. Further studies could reveal how these two Rabs on the same donor membrane site regulate vesicles (possibly with different cargoes) targeting different acceptor membranes. Supplementary data The following supplementary data are available at JXB online. Fig. S1. Phenotypes of phytoene desaturase (PDS) (positive control), Luciferase (Luc) (negative control) and NbRabF1 knock-down plants. Fig. S2. Isolation of His-tagged NbRabF1 from E. coli analyzed via SDS-PAGE (A), and in vitro GTPase activity assay of NbRAF1 (B). Acknowledgements We appreciate the Bioimage Core Laboratory of the Graduate Institute of Biotechnology at National Chung Hsing University, Taichung, Taiwan for providing facilities and the assistance. This work was financially supported (in part) by the Advanced Plant Biotechnology Center from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) and grants from the Ministry of Science and Technology in Taiwan (MOST 107-2313-B-320-001-MY3 and 106-2311-B-005-003-MY3). 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Published by Oxford University Press on behalf of the Society for Experimental Biology.
TI - Dissecting the role of a plant-specific Rab5 small GTPase NbRabF1 in Bamboo mosaic virus infection
JO - Journal of Experimental Botany
DO - 10.1093/jxb/eraa422
DA - 2020-12-31
UR - https://www.deepdyve.com/lp/oxford-university-press/dissecting-the-role-of-a-plant-specific-rab5-small-gtpase-nbrabf1-in-AGwGXL8PS0
SP - 6932
EP - 6944
VL - 71
IS - 22
DP - DeepDyve
ER -