Fig 1.
Presentation of the CaMV infection cycle and the split GFP system.
(A) The circular double-stranded DNA genome (~8 kbp; circle with arrows) of CaMV is encapsidated in an icosahedral virus particle (mauve hexagon) and codes for six proteins (P1-P6, arrows) that are detected in infected plants. The GFP11 tag (grey box with green border) is fused to the P6 coding sequence yielding 11P6. (B) The infection cycle starts with virus particles (VPs) being delivered into the cytoplasm of a plant cell after it has been punctured by the stylets of an aphid vector. VPs dock at the nuclear envelope and disassemble to allow the naked viral DNA to enter the nucleus. There, the viral genome is transcribed to produce two mRNAs, the 19S RNA encoding P6, and the pregenomic, polycistronic 35S RNA encoding also the other viral proteins. P6 belongs to the early proteins that are translated in the cytoplasm (note that P6 has been replaced by 11P6 in this study). Within the cytoplasm, P6 accumulates in foci that will give rise to the virus factories [here is exemplified one (VF)] with P6 forming the matrix protein, where all viral synthesis occurs and most progeny VPs are stored. Viral synthesis in the VFs involves many coordinated events including the P6-mediated translation transactivation required for the translation of all viral proteins from the polycistronic 35S RNA. The translation products include P1 or MP, the movement protein that associates with the plasmodesmata and is required for cell-to-cell and systemic movement of the virus; P2 or ATF, the aphid transmission factor that binds the virus particles to the aphid vector mouthparts during plant-to-plant transmission; P3 or VAP, the virus-associated protein, P4 or CP (capsid protein), and P5 or RT, the reverse transcriptase generating progeny DNA genomes from the 35S RNA. P6 or TAV (transactivator-viroplasmin) is, besides a transactivator and VF matrix protein, an RNA silencing suppressor that interferes with specific anti-viral defense pathways. Because CaMV engineered to express 11P6 is infectious (as demonstrated in this study), 11P6 is presumed to be functional in all the above stated P6 activities. Besides VFs, a second type of viral inclusions, the transmission bodies (TBs), forms during infection. TBs contain P2, P3 and some VPs and are entirely dedicated to aphid transmission. (C) The split GFP system used in this study. The transgenic reporter plant (top left) expresses the non-fluorescent GFP1-10 (gray barrel with green outline). When infected with CaMV11P6, 11P6 produced during infection associates with GFP1-10, yielding fluorescent GFP1-10/11P6 complexes (green barrel) that can be observed by real time fluorescence microscopy or macroscopy. The aphid drawing in (A) is from [24].
Fig 2.
Symptom development on CaMV-infected plants and stability of the insertion.
(A) Arabidopsis thaliana GFP1-10 plants 31 days and (B) turnip plants 36 days after mechanical inoculation with plant extracts prepared from infected (CaMVwt or CaMV11P6) or non-infected plants as indicated. Scale bars are 5 cm. (C) Close-up showing attenuated symptoms of turnip leaves of CaMV11P6-infected plants compared to CaMVwt-infected plants at 27 dpi. For comparison, leaf tissue from a non-infected plant is shown as well. (D) The genomic region encompassing the 11GFP tag was amplified by PCR from total extracts prepared from plants infected with CaMVwt or CaMV11P6 as indicated. The viruses had been passaged serially several times (>10 times for A. thaliana and >4 times for turnip) before the experiment. To determine whether the viral DNA was encapsidated, extracts were (+) or were not (-) incubated with DNase before PCR to digest free DNA. To verify efficiency of the DNase treatment, extracts from mock-inoculated turnip leaf were spiked with CaMV encoding plasmid DNA before DNase treatment (Mock). Amplification of CaMVwt DNA yielded a 655 bp product, amplification of CaMV11P6 DNA a 721 bp product.
Table 1.
Aphid transmission rate of CaMV11P6.
Fig 3.
Kinetics of viral protein accumulation in turnip leaves.
Total extracts of samples taken from the same systemically infected leaf at the time points indicated were analyzed by Western blotting with antisera against P2, P3, P4 or P6 (indicated by red arrows). The first panel (RuBisCO) shows a loading control (Ponceau Red staining of the large chain RuBisCO subunit). It should be noted that under the electrophoresis conditions used, much of the capsid protein P4 did not enter the gel properly and was retained in the upper part of the gels (blue arrow). The three red arrows in the anti-P4 blot point to the various mature P4 forms detected in infected plants [25]. Shown are the representative Western blots from one experiment out of seven performed.
Fig 4.
Visualization of 11P6 in CaMV11P6-infected A. thaliana GFP1-10 plants.
(A) GFP1-10 plants were mechanically inoculated at different times with CaMV11P6 and analyzed at the indicated day post inoculation (dpi) for GFP fluorescence with a fluorescence scanner. The figure is a collage from different acquisitions and plants are presented at different magnification scales. Two plants are presented for 28 dpi and 36 dpi to show different infection states. Non-infected and CaMVwt-inoculated (21 dpi) plants are included as negative controls. (B) 11P6 fluorescence is observed before appearance of visual symptoms. Arabidopsis thaliana GFP1-10 plants were mechanically inoculated with CaMV11P6 and leaves analyzed at 24 dpi for GFP fluorescence and visual symptoms with a G:Box (upper panel). The leaves were also analyzed for symptoms with a color camera (lower panel). The three images present 11P6 fluorescence in symptomatic and unsymptomatic leaves. The various leaves in the image to the right are from the same plant. Note that the leaves indicated by the yellow arrowheads are not yet infected. (C) The two pictures show negative controls where either CaMV11P6 was inoculated in Col0 plants or CaMVwt in GFP1-10 plants as indicated. Images were acquired at 32 dpi with a G:Box or a color camera as described above.
Fig 5.
Microscopic analysis of CaMV11P6-infected A. thaliana.
(A) Arabidopsis thaliana GFP1-10 leaves were analyzed by confocal fluorescence microscopy at 3 and 40 days after inoculation (dpi) with CaMV11P6. GFP fluorescence and chloroplast autofluorescence are presented in green and grey, respectively. The greenish spots in the leaf tissue at 3 dpi are not due to GFP since they also fluoresced in blue when excited at 405 nm. The inset in the third panel shows details of an 11P6 inclusion in which darker circular spots are visible (yellow arrows). (B) CaMVwt-infected tissue sections were immunolabeled (magenta) at 28 dpi using the antisera (αP2 or αP6) as indicated. The yellow arrows point to the stronger stained cortex of immunostained VFs, the inset presents details of a VF. (C) CaMV11P6-infected tissue was immunolabeled at 40 dpi using P2 antiserum (magenta) and 11P6 was visualized by fluorescence of the reconstituted split GFP (green). The inset shows a confocal single section of such a green fluorescing inclusion labeled with P6 antiserum (magenta). Note that only the cortex of the inclusion is labeled. The yellow arrows point to putative VF lacunae. Cell walls in (B) and (C) were stained with Fluorescent Brightener 28 and are presented in blue. All confocal images are maximum projections except where indicated. (D) and (E) Transmission electron micrographs presenting (D) a CaMVwt and (E) a CaMV11P6-infected cell, both fixed at 33 dpi. VF and TB designate virus factories and transmission bodies, respectively; yellow lines (labeled “VP”) and blue arrows point to virus particles and lacunae, respectively. Different microscopes were used for image acquisition in (D) and (E). Brightness and contrast were corrected to allow better comparison of the micrographs. Scale bars in (A) 50 μm for the overviews and 5 μm for the inset, in (B) and (C) 10 μm and in (D) and (E) 1 μm.
Fig 6.
Observation of CaMV11P6-transfected A. thaliana GFP1-10 protoplasts.
Protoplasts were transfected with infectious CaMV11P6 plasmid (A) or with CaMV11P6 virus particles (B), and observed 15 h later by confocal fluorescence microscopy. Protoplasts display fluorescent 11P6 foci (green spots indicated by yellow arrows) and weak cytosolic 11P6 label (blue arrow). The images shown in (A) or (B) are from the same protoplast. The left panels present optical single sections, the right panels maximum projections, and the middle panels bright field illumination images. Chloroplasts are presented in magenta. Scale bars in all panels represent 10 μm.
Fig 7.
Cell lysates, prepared from non-infected controls (non-inf.) or CaMV11P6-infected A. thaliana GFP1-10 plants (11P6), were incubated with magnetic beads with immobilized anti-GFP nanobodies. Total cell extracts (Total) or proteins retained on the beads (Beads) were separated on different gels by SDS-PAGE and analyzed by Coomassie Blue staining or Western blotting. (A) Coomassie blue staining reveals proteins with molecular masses of approximately 65 kD and 25 kD from CaMV11P6-infected but not from healthy control lysate. The weakly stained 55 kD and 15 kD proteins visible in the healthy control probably represent carried-over small and large chain RuBisCO subunits, respectively. (B) Membranes were cut in two and revealed for P6 (upper blots) or GFP (lower blots). This allowed to identify the 65 kD protein as 11P6 and the 25 kD protein as GFP1-10.