Materials and Reagents

3-4 weeks old Cucumis melo L. subsp. melo cv. Galia plants
Agrobacterium tumefaciens (A. tumefaciens) strain C58C1, or similar, transformed with the binary vector harboring the GFP-tagged viral genome
In our case, pMNSV(Al)/GFP encoding GFP-tagged Melon necrotic spot virus (MNSV) genome under the control of the Cauliflower mosaic virus (CaMV) 35S promoter and the potato proteinase inhibitor terminator (PoPit) (Figure 1).

Figure 1. Schematic representation of the recombinant infectious clone pMNSV(Al)/GFP used in this assay
Yeast extract (Difco, catalog number: 212750 )
Tryptone (Difco, catalog number: 211705 )
Sodium chloride (NaCl) (Panreac Applichem, catalog number: 131659 )
Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
MES (Sigma-Aldrich, catalog number: M8250 )
Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M9272 )
Antibiotics
LB medium (see Recipes)
Agrobacterium infiltration buffer (see Recipes)

Equipment

28 °C growing chamber
15 ml culture tubes
Swinging centrifuge rotor for 15 ml tubes
1.5 ml tubes (standard Eppendorf tubes or similar)
BioPhotometer plus (Eppendorf, catalog number: 6132000008 )
1 ml syringes without needle
Plant growing chamber
Leica MZ12 fluorescent stereo microscope

Software

Adobe Photoshop CS5 or higher
ImageJ, FIJI or similar software
MS Excel

Procedure

Viral inoculation by agro-infiltration
1. Inoculate a single colony of A. tumefaciens transformed with pMNSV(Al)/GFP in 5 ml of LB medium with Kanamycin (50 µg/ml) and Rifampicin (50 µg/ml). Incubate the culture overnight (about 12-16 h) at 28-30 °C with vigorous shaking.
2. Measure the A₆₀₀ of the culture.
3. Collect the bacteria by slow-speed centrifugation (2,000 x g, 15 min) in a swinging rotor. Remove the supernatant.
4. Resuspend the pellet with Agrobacterium infiltration buffer. Since some viruses replicate and then move faster than others, the final A₆₀₀ need to be adjusted depending on the virus. Large inoculum of a fast-moving virus may generate a high number of viral infection foci so close between them that they could be quickly merged. Therefore the resulting average size of the infection foci could be overestimated. If this occurs, reduce the final A₆₀₀ until you get infection foci physically separated from each other. If, instead, only few infection foci are observed, the final A₆₀₀must be increased.
5. Leave at room temperature for 2 h before infiltration.
6. Infiltrate the cultures with 1 ml syringe into the lower side of the leaves. Use 3-4 week-old Cucumis melo plants and avoid non fully-expanded leaves. Use your fingertip to apply gentle counter pressure to the other side of the leaf. A fully infiltrated part of the leaf gives a water-soaked appearance.
7. Label de infiltrated leaves with a ring of adhesive one-sided tape placed around the petiole.
8. Keep the plants in growth chambers in 16 h light at 25 °C and 8 h dark at 22 °C.
Image acquisition
1. Several days after infiltration check the appearance of fluorescent infection foci with a Leica MZ12 fluorescent stereo microscope or similar device. The exact time will depend on which virus is assayed (5-8 days after infiltration).
2. Place the melon leaves beneath the objective (Planachromatic 0.5x) and engage the lowest magnification factor (0.71x)
3. Select GFP1 filter set [excitation filter 425/60 nm (395-455 nm), barrier filter 480 nm].
4. Look into the eyepieces and bring the fluorescent foci into focus using the rotatory knob.
5. Correct the values for "exposure", "gain" and "gamma" until you obtain the desired results for the image and capture it. Don’t forget to add a scale bar.
Image analysis
1. Open the image with Adobe Photoshop CS5 or higher.
2. If necessary, enhance the image brightness and contrast until infection foci have well-defined borders.
3. Use the magic wand selection tool to select the infection foci. This tool selects similarly colored non-contiguous areas. If the magic wand selects less area than you want, increase the tolerance setting.
4. Fill selection with black color by selecting “Fill” in the “Edit” menu.
5. Invert selection by choosing “Invert” in the “Select” menu and fill with white color. If you have infection foci that have merged together separate them by drawing a white line were it feels the division should be. Save the resulting black and white image (Figure 2).
  
  Figure 2. Images of after B and before A the Adobe Photoshop editing
6. Open the black and white image with ImageJ.
7. Select “Binary” in the “Process” menu and “Make binary”.
8. Check the spatial calibration of the image. If the image is calibrated, the number of pixels in each dimension and dimensions with a unit of length will be visible at the top of the image window. If the image is not calibrated, only the number of pixels will be written and you will need to set the dimension of the image manually. Then, open an image with a scale bar and using the "Straight line" tool, draw a line along the scale bar. In the “Analyze” menu select “Set scale”. A dialog box will open and the distance in pixels will be automatically adjusted. By choosing global, you set this calibration for all images you open during the ImageJ session.
9. Select “Analyze particles” in the “Analyze” menu. In the new dialog box, set size range by trial and error to exclude tiny background particles. Choose “Exclude on edges” option to avoid counting partial infection foci at the edges of the image. Check “Include holes” if you want it to fill in holes and include that area in the measurement. Under “Show” select “Overlay outlines” to number the infection foci that are measured. These numbers correspond to data for individual particles that will be listed in the “Results” window if you check the “Display results” and click “Ok”.
10. Save results as MS Excel file and calculate the average size of infection foci in MS Excel.

Recipes

LB medium
Mix 5 g of yeast extract with 10 g of tryptone, 10 g of NaCl
Add dH₂O to 1,000 ml
Agrobacterium infiltration buffer
10 mM MES (pH 5.6)
10 mM MgCl₂
150 µM acetosyringone

Acknowledgments

This work was funded by grant BIO2011-25018 from the Spanish Ministerio de Economia y Competitividad and by Prometeo Program GV2011/003 from the Generalitat Valenciana. J.A.N. and M.S. are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Educacion y Ciencia of Spain.

References

Kim, J. H., Lee, S. R., Li, L. H., Park, H. J., Park, J. H., Lee, K. Y., Kim, M. K., Shin, B. A. and Choi, S. Y. (2011). High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6(4): e18556.
Serra-Soriano, M., Pallas, V. and Navarro, J. A. (2014). A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements. Plant J 77(6): 863-879.