Localizing Genome Segments and Protein Products of a Multipartite Virus in Host Plant Cells

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successfully used either separately or in combination in a number of studies. Generally, in situ hybridization protocols include numerous steps, resulting in a cumbersome and time-consuming technique. The protocol developed by Ghanim et al. has considerably simplified this technique . When applied to our pathosystem (FBNSV, Vicia faba), however, this protocol yielded poor and/or non-specific signals. Although FBNSV is a circular single-stranded DNA phytovirus like the Tomato yellow leaf curl virus (TYLCV) on which Ghanim and coauthors tested their protocol, the FBNSV genome is composed of eight molecules of around 1 kb each, sharing common regions which sometimes correspond to the almost quasi-totality of their non-coding regions (Grigoras et al., 2010).
We thus sought to improve this protocol for the detection of individual FBNSV genomic segments by using, instead of fluorescent oligonucleotides, random primed probes generally used for in situ localization experiments on chromosomes. We were able to couple it with immunolocalization for visualization of nucleic acid and proteins i.e., covisualization of genetic information and translation products within the same cells.     (Table 1) corresponding to the coding regions of each genomic segments were amplified by PCR using the GoTaq Polymerase kit (Promega) and the conditions summarized in Table 2. PCR products were then run on a 1% agarose gel and gel purified using the Wizard SV Gel and PCR Clean-Up System (Promega). These amplicons were then used as templates to produce segment-specific probes. DNA labeling was performed using the Bioprime DNA labeling b. And distilled Water to a total volume of 49 μl.

Mix briefly by vortexing or pipetting.
4. Add 1 μl of Klenow Fragment. Mix gently but thoroughly. Centrifuge at maximum speed (no specific recommendation is included in the manufacturer notice, when we proceed to it, we do it at maximum speed of a bench centrifuge) for 15-30 s.

Add 5 μl of Stop Buffer and mix.
The probes can be stored at -20 °C for months. The probes chosen must be specific of the DNA or RNA sequence you want to localize. The specificity of the probe can be checked following the procedure detailed in the next section.    6. Incubate for at least 2 h at 37 °C on a rotator. This incubation step can also be done overnight.
7. Rinse the membranes three times using hybridization buffer, for 5 min each, and once with PBS 1x for 5 min. Do the rinse steps at room temperature. 8. Dry the membranes with Whatman paper. 9. Results can be visualized using a phosphorimager.    Parameters were adjusted to obtain sufficient resolution and fluorescence intensity signal recoveryin a chosen series of infected plant exhibiting a high intensity of fluorescence-without saturation points. Once these parameters were set, all of the images were acquired using the same parameters so that they could easily be compared with one another.
In our case, images were taken with a 40x water immersion objective with a resolution of at least 512 x 512 and with a pinhole aperture of 1 Airy Unit so as to work in confocal mode.
We set on a configuration with 3 sequential tracks, one for each fluorochrome used.

Data analysis
Analyses were run using maximum intensity projections so that all the fluorescence emitted in the whole nuclei was accounted for. These microscopic images were analyzed using Image J software.
1. First of all, in order to minimize the signal caused by the autofluorescence of the tissues-either natural or induced by fixation, grey level intensities coming from the green and red tracks were decreased independently on each photo until no green or red fluorescence could be visually detected in tissues where the FBNSV is known to be absent (i.e., in xylem and mesophyll; the FBNSV being a phloem restricted virus). This computer process was done in the exact same way for all pictures and applied equally to the whole surface of the image ( Figure 6).

Note: The aim of this step was to simply distinguish infected cells from non-infected ones with
a high level of background autofluorescence. As a consequence, only cells in which green or red fluorescence levels exceeded the level of background fluorescence were considered for further analysis. As we were mainly interested in the cells in which our ssDNA virus can replicate, we focused our analyses on phloem nucleated cells; nuclei stained using DAPI. 2. On the images, we manually delineated the DAPI stained area (corresponding to the nucleus in each cell) using a cursor and quantified intensity of grey level values for each pixel using the ImageJ software. The intensity of grey level was also determined for both green and red fluorescence.
3. We then calculated the average pixel fluorescence intensity value for each nucleus and each fluorochrome. We thus obtained an average intensity value (in arbitrary fluorescence unit) for both green and red fluorescence for each selected individual cell within an infected petiole (the FBNSV replicate and accumulate in the nucleus of infected cells). 4. In order to check the reliability of our analysis, we produced two probes respectively targeting the two moieties of the same segment R: "probe-R1" and "probe-R2", one labeled green and the other red. In such a control experiment, we could confirm that absolutely all nuclei with a significant red signal also had a significant green signal. When quantifying these green and red fluorescence, we observed a highly significant correlation, logically showing that the accumulation of one part of a genome segment within a cell is clearly correlated to that of the other part of the same segment. Around 50 infected cells were analyzed by condition.
5. Images acquired from the same slide with different resolutions (512 x 512 and 1024 x 1024) and at different times post labeling (2 weeks between the two-time points) were analyzed and led to the same results. 6. Linear regression analyses were performed with the JMP software.