The pSC1001a vector containing the iLOV sequence was a kind gift from S. Chapman (The James Hutton Institute, Dundee, Scotland). The PVX-based vector pPVX-iLOV-2A-CP_{PV X} was constructed by amplifying the iLOV sequence using specific primers NheI-iLOV and iLOV-BspEI, which also introduced the named restriction sites (Table 1). The PCR products were inserted into the pCR2.1-TOPO cloning vector and introduced into competent Escherichia coli TOP10 cells according to the manufacturer’s recommendations (Thermo Fisher Scientific, Karlsruhe, Germany). A control PCR using the M13 forward and reverse primers was carried out to confirm the presence of the target gene. The iLOV sequence was digested with NheI and BspEI and transferred to vector pTCXIIc (Shukla et al., 2014) which was linearized with the same enzymes to remove the mCherry sequence, leaving the FMDV 2A sequence as a ligation target. The integrity of the final plasmid was confirmed by PCR using primers TGB-fw and CX1.

The TMV-based vectors containing the iLOV sequence were constructed by splice overlap extension (SOE) PCR. The TMV cp sequence was amplified from vector pET22b-TMVCP-His (unpublished data) using primers PacI-CPTMV and CPTMV-rev or PacI-CPTMV and CPTMV-G4S, whereas the iLOV sequence was amplified from vector pPVX-iLOV-2A-CP_{PV X} using primers CPTMV-iLOV and iLOV-Stop-NotI or G4S-iLOV and iLOV-Stop-NotI. The primer combinations were chosen according to whether or not the linker sequence was required. The resulting CP and iLOV fragments were combined and re-amplified with flanking primers in two subsequent PCRs. The CP_TMV-iLOV and CP_TMV-G₄S-iLOV fusion sequences were inserted into vector pCR2.1-TOPO for the transformation of competent E. coli TOP10 cells as described above. The integrity of the vectors was confirmed by PCR using the M13 forward and reverse primers. The pCR2.1 CP_TMV-iLOV and pCR2.1 CP_TMV-G₄S-iLOV vectors were digested with PacI and NotI and the inserts were transferred to vector pTRBOc (Lindbo, 2007) which had been linearized with the same enzymes, thus fusing the iLOV sequence to the C-terminus of the TMV CP either directly or via the (G₄S)₃ linker. The integrity of the final vectors pTMV-CP_TMV-iLOV and pTMV-CP_TMV-G₄S-iLOV was confirmed by PCR using primers TMV5482f and TMV6269r.

Vector pTMV-iLOV-2A-CP_TMV was constructed by amplifying the iLOV sequence from pPVX-iLOV-2A-CP_{PV X} using primers PacI-iLOV and 2A-rev, whereas the TMV CP sequence was amplified using primers N-2A-CPTMV and CPTMV-Stop-NotI. The fragments were joined by SOE-PCR, re-amplified and transferred to vector pTRBOc as described above to produce final vector pTMV-iLOV-2A-CP_TMV. The integrity of the vector was determined by PCR as described above.

The subgenomic expression vector was produced by amplifying the iLOV sequence using primers PacI-iLOV and iLOV-Stop-NotI. The product was transferred to vector pCR2.1-TOPO and its integrity was confirmed as described above, and then the insert was excised with PacI and NotI and transferred to vector pJL (Lindbo, 2007), which had been linearized with the same enzymes. The integrity of the resulting vector pJL-iLOV was confirmed by PCR using primers TMV5482f and TMV6269r.

In TMV-derived viral vectors, the expression of the proteins of interest is controlled by the CP subgenomic promoters. All vectors were maintained and amplified in the E. coli strain DH5α and their integrity was confirmed by sequencing prior to the inoculation of N. benthamiana plants. Full sequences of the recombinant viruses can be found online as Supplementary Material.

Three leaves from 4-week-old N. benthamiana plants were each inoculated with 10 μg of pTMV-CP_TMV-iLOV, pTMV-CP_TMV-G₄S-iLOV, pTMV-iLOV-2A-CP_TMV, pJL-iLOV, or pPVX-iLOV-2A-CP_{PV X}. The leaf surface was gently abraded with Celite 545. After incubation for 15–20 min, the leaves were rinsed with water to remove Celite and excess DNA. The plants were cultivated in a phytochamber (12-h photoperiod, 5000–10,000 lux, 26°C/20°C light/dark temperature, humidity 70%).

SDS-PAGE was carried out using 12% resolving gels and a 4% stacking gels loaded with 20 μl of sample per lane. Samples for SDS-PAGE followed by western blot were prepared by extracting total protein from systemically infected leaves 1:2 in PBS, followed by centrifugation (20,000 × g, 15 min, 4°C) and boiling for 5 min in 5× reducing loading buffer (Laemmli, 1970). For in-gel fluorescence detection by UV light, the samples were prepared as above but were not boiled. The separated proteins were either stained with Coomassie Brilliant Blue G250 or transferred onto a Hybond C nitrocellulose membrane (GE Healthcare, Munich, Germany) using the semi-dry blotting system (BioRad, Munich, Germany). The membrane was blocked for at least 30 min in 4% skimmed milk in PBS. The iLOV protein was detected at room temperature overnight using an iLOV-specific polyclonal antibody (kindly provided by John Christie, Institute of Molecular Cell and Systems Biology, University of Glasgow, Scotland) diluted 1:4000 in PBS, followed by incubation for at least 2 h with a secondary alkaline phosphatase-conjugated goat-anti-rabbit (GAR^AP) antibody (Dianova, Hamburg, Germany) diluted 1:5000 in PBS. The TMV and PVX were detected using a TMV-specific polyclonal antibody (Bioreba, Reinach, Switzerland) or a PVX-specific polyclonal antibody (DSMZ, Braunschweig, Germany) each diluted 1:5000 in PBS, followed by the secondary GAR^AP antibody as described above. The signals were visualized with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyphosphate (NBT/BCIP) p-toluidine salt (Roth, Karlsruhe, Germany). The P7712 Pre-stained Protein Standard (New England Biolabs, Ipswich, Massachusetts, USA) was used for sizing. We estimated the ratio of fusion protein and free iLOV by densitometric analysis of the αiLOV western blot by using ImageJ v1.50f software. The value of the iLOV-2A-CP_TMV band was divided by the density values of all bands for the fusion protein and free iLOV, multiplied by 100.

For the sequences analysis of progeny viruses, total RNA from systemically infected N. benthamiana leaves was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 2 μg RNA samples were treated with 3 U of DNaseI (Thermo Fisher Scientific, Waltham, United States) prior to cDNA synthesis by M-MLV Reverse Transcriptase RNaseH Minus Point Mutant (Promega, Madison, WI, United States). 1 μg DNase-digested RNA was mixed with 0.5 μl TMV6269r primer, incubated for 10 min at 80°C and for 10 min at 4°C allowing primer annealing. For cDNA synthesis 5 μl 5x M-MLV reaction buffer, 1 mM dNTPs, 2.5 μl DEPC-H₂O, and 1 μl M-MLV reverse were added and reverse transcription was carried out (30 min at 40°C, 20 min at 45°C, 20 min at 50°C, 20 min at 55°C, 20 min at 70°C). The integrity of CP_TMV-iLOV, CP_TMV-G₄S-iLOV, iLOV-2A-CP, and iLOV was confirmed by PCR using the TMV5482f and TMV6269r primers amplifying a region between the movement protein and the CP_TMV coding sequence. The products were resolved by 1.2% agarose gel electrophoresis in 1x TAE buffer. Ethidium bromide was used to visualize the DNA under UV light.

Pioloform-coated nickel grids (Plano, Wetzlar, Germany) were used for all preparations. For immunogold decoration the grids were incubated on a drop of systemically infected plant leaf extract for 30 min at room temperature, washed once with 20 drops of PBS containing 0.1% Tween-20 (PBST), and blocked with 0.5% bovine serum albumin (Sigma, Taufkirchen, Germany) in PBS for 20 min. After blocking, adsorbed hybrid particles were incubated for at least 2 h with the iLOV-specific antiserum described above (diluted 1:100), washed again and the particles were detected with a goat-anti-rabbit antibody conjugated to 15-nm gold particles (British BioCell, Cardiff, United Kingdom) for at least 2 h. Before staining with five drops of 1% uranyl acetate, the grids were extensively washed with PBST and twice with water. Transmission electron microscopy was carried out using a Zeiss EM10 microscope (Carl Zeiss AG, Jena, Germany).

Hybrid particles were captured from systemically infected leaf extracts by an αiLOV antibody. Therefore, the grids were incubated for 30 min on the iLOV-specific antibody, before washing and blocking with 0.5% BSA as described above. After another washing step the antibody was used to capture the TMV-iLOV particles from leaf extracts. The grids were washed and stained as described above.

We constructed three TMV-based vectors (Figure 1) in which the iLOV coding sequence was joined to the 3′ end of the TMV cp gene (a) directly or (b) via a glycine-serine linker. Furthermore, the sequence was fused with (c) a FMDV 2A sequence to the 5′ end of the cp gene to enable a ribosomal skip during translation. A fourth TMV-based vector with a CP subgenomic promoter was used to express the free iLOV protein. As a positive control the iLOV sequence was also fused to the 5′ end of the PVX CP via the FMDV 2A sequence.

The vectors were used to inoculate Nicotiana benthamiana plants, resulting in systemic infections in all cases as revealed by the green fluorescence of the iLOV polypeptide, even in plants inoculated with pTMV-CP_TMV-iLOV and pTMV-CP_TMV-G₄S-iLOV (Figure 2). However, long-distant movement was delayed in plants infected with pTMV-CP_TMV-iLOV and pTMV-CP_TMV-G₄S-iLOV, and the fluorescence signal was therefore present mostly in the stem whereas only small portions of leaf tissue showed iLOV fluorescence 21–29 dpi (Figures 2B,C). Plants infected with pTMV-iLOV-2A-CP_TMV and pPVX-iLOV-2A-CP_{PV X} showed systemic fluorescent spots from 6 to 7 dpi onward. In addition to the bright green fluorescence, the plants inoculated with pTMV-iLOV-2A-CP_TMV also showed severe symptoms of TMV infection, including mosaic-like mottling, dwarfing and necrosis at 11 dpi (Figure 2D). The necrotic lesions were more severe when the free iLOV polypeptide was expressed from the subgenomic promoter (Figure 2F).

Leaves were collected from plants systemically infected with pTMV-CP_TMV-iLOV, pTMV-CP_TMV-G₄S-iLOV, pTMV-iLOV-2A-CP_TMV, pJL-iLOV, and pPVX-iLOV-2A-CP_{PV X}, and the modified CPs were characterized by SDS-PAGE and western blotting using antibodies specific for TMV or iLOV.

Western blots of total leaf extracts from plants infected with pTMV-CP_TMV-iLOV and pTMV-CP_TMV-G₄S-iLOV showed only faint bands at the anticipated molecular weights of 30.5 and 31.4 kDa when probed with the TMV-specific antibody (Figure 3B) but strong signals when probed with the iLOV-specific antibody (Figure 3C), probably due to epitope access and weak systemic leaf infection. The TMV-specific antibody also revealed a 17.6 kDa band corresponding to the free TMV CP (Figure 3B) but as expected we were unable to detect the free iLOV polypeptide, either by western blot or under UV light. Weak bands corresponding to the CP_TMV-iLOV and CP_TMV-G₄S-iLOV fusion proteins were also revealed under UV light. The weakness of the signal was probably due to the high background caused by the autofluorescence of the leaf extracts (Figure 3D).

Western blots of total leaf extracts from plants infected with pTMV-iLOV-2A-CP_TMV showed only a faint band at the anticipated molecular weight of 32.5 kDa when probed with the TMV-specific antibody (Figure 3B) or with the iLOV-specific antibody (Figure 3C). As above, the TMV-specific antibody also detected a 17.6 kDa band corresponding to the free TMV CP. However, in this case the iLOV-specific antibody detected not only the fusion protein but also the 13 kDa free iLOV polypeptide (Figure 3B). Only the latter could be detected under UV light (Figure 3D). Densitometric analyses show a ratio of 2:3 for iLOV-2A-CP_TMV fusion protein to free iLOV.

The TMV vector, plants infected with pPVX-iLOV-2A-CP_{PV X} displayed a strong signal for the fusion protein (39.9 kDa) in western blots probed with either the PVX-specific or iLOV-specific antibodies (Figures 3E,C). A 25 kDa band also detected by western blot with the PVX-specific antibody represented the free PVX CP (Figure 3E). The 13 kDa free iLOV polypeptide was detected by western blot using the iLOV-specific antibody (Figure 3C) and also produced a strong signal under UV light (Figure 3D).

Leaf extracts from plants infected with the TMV subgenomic promoter control construct yielded strong signals for both the free TMV CP and the free iLOV polypeptide when detected with the corresponding antibodies (Figures 3B,C), and the iLOV polypeptide was also detected under UV light as expected (Figure 3D).

Transmission electron microscopy confirmed the presence of iLOV on the surface of CP_TMV-iLOV (Figure 4B), CP_TMV-G₄S-iLOV (Figure 4C), iLOV-2A-CP_TMV (Figure 4D), and iLOV-2A-CP_{PV X} particles (Figure 4G) due to the recognition of the iLOV polypeptide by specific antibodies conjugated to 15-nm gold particles. In contrast to the iLOV-2A-CP_TMV and iLOV-2A-CP_{PV X} particles, only a few decorated TMV particles were observed in plants expressing CP_TMV-iLOV and CP_TMV-G₄S-iLOV. Moreover, the TMV-iLOV particles were captured from leaf extracts by the iLOV-specific antibody (Figures 4I–K). Although particles were not decorated in plants infected with pJL-iLOV expressing the free iLOV polypeptide from a subgenomic promoter (Figure 4E), some particles were captured by the iLOV antibody (Figure 4L), and were also detected in the western blot probed with this antibody (Figure 3C). TMV and PVX particles without fusion proteins were not decorated by the antibodies as expected (Figures 4F,H).

Total RNA was isolated from N. benthamiana leaves systemically infected with pTMV-CP_TMV-iLOV, pTMV-CP_TMV-G₄S-iLOV, pTMV-iLOV-2A-CP_TMV, or pJL-iLOV. Non-infected leaves served as negative controls. Residual RNA was removed by RNase prior to reverse transcription of the viral RNA with a TMV specific primer. The cDNA and control plasmids were amplified by primers flanking a region between the movement protein and cp genes (Figure 5). pTMV-CP_TMV-iLOV and pTMV-CP_TMV-G₄S-iLOV infected leaves should have yield fragments of 1216 and 1261-bp in size, but revealed only deleted versions with a size of ∼900 bp. Infections with pTMV-iLOV-2A-CP_TMV and pJL-iLOV indicated products of 1276 or 733 bp in RT-PCR analysis, as expected.