Detection of Poly (A) RNA in Mesophyll Cells of Nicotiana benthamiana Using in situ Hybridization

Materials and Reagents

1. Leaves of 3-4 weeks old wild type or Nup gene silenced N. benthamiana
   Notes:
   a.This method can be applied for other dicot plants with relatively soft leaves.
   b.For Virus-induced gene silencing (VIGS) of N. benthamiana. See Zhang and Liu (2014).
   
2. 10 µM 48-mer oligo d(T) 5’-labeled with Alexa Fluor-488, HPLC-purified [purchased from custom oligo services (e.g. Eurofins Genomics)]
   Note: Dissolved in TE (Tris-EDTA) buffer and store at -80 °C, shaded.
   
3. 99.8% Methanol (4 ml per sample) (Wako pure chemical, special glade, catalog number: 131-01826 )
   
4. 99.5% Ethanol (5 ml per sampl) (Wako pure chemical, special glade, catalog number: 057-00456 )
   
5. 99.5% Ethanol/Xylene (1:1 v/v, 1 ml per sample) (Wako pure chemical, special glade, catalog number: 244-00086 )
   
6. 99.8%Methanol/Fixation solutionB (1:1 v/v, 1 ml per sample)
   
7. PerfectHyb Plus Hybridization Buffer (2 ml per sample) (Sigma-Aldrich, catalog number: H7033 )
   
8. Fixation cocktail (see Recipes)
   
9. Fixation solution A (see Recipes)
   
10. Fixation solution B (see Recipes)

Equipment

1. 3 ml vial containers
   
2. Rotary shaker (e.g. BioCraft, model: BC-730 )
   
3. Hybridization oven or incubator (50 °C) with rotary shaker (e.g. TAITEC, model: BR-23FP MR )
   
4. Confocal laser-scanning microscope (preferred) or ordinary fluorescent microscope with the filter set for GFP or Alexa Fluor-488 (e.g. Olympus, model: FV1000-D )

Procedure

1. Poly (A) RNA in situ hybridization
   
   1. Cut leaves of N. benthamiana into small pieces (e.g. 3 mm x 3 mm) by knife and put ~10 leaf pieces/sample in 3 ml vial container.
      Note that the center of larger pieces won’t be stained with AF488 probe.
      
   2. Add 1 ml Fixation solution A in the vial container and shake (Approx. 80 rpm) for 30 min at room temperature (RT). Leaf pieces will float on the surface of the solution at the start, but will sink later (Figure 1).
      
       
      Figure 1. Leaf discs of N. benthamiana in Fixation solution A before (left) and after (right) shaking for 30 min (step A2)
      
      
   3. Remove Fixation solution A by pipeting.
      
   4. Add 1 ml methanol. Shake (80 rpm) for 5 min at RT and remove methanol (twice). Note that the inner side of the vial should be washed well with methanol.
      
   5. Add 1 ml ethanol, shake (80 rpm) for 5 min at RT and remove ethanol (three times).
      
   6. Add 1 ml ethanol/xylene (1:1). Shake (80 rpm) for 30 min at RT and remove the solution. Note that leaf tissues will become white during steps A4-6.
      
   7. Add 1 ml 100% ethanol. Shake (80 rpm) for 5 min at RT and remove ethanol (twice).
      
   8. Add 1 ml 100% methanol. Shake (80 rpm) for 5 min at RT and remove ethanol (twice).
      
   9. Add methanol/Fixation solution B (1:1). Shake (80 rpm) for 5 min at RT and remove the solution.
      
   10. Add 1 ml Fixation solution A and shake (80 rpm) for 30 min at RT.
       
   11. Remove Fixation solution A.
       
   12. Add 1 ml Fixation solution B. Shake (80 rpm) for 5 min at RT and remove the solution (twice).
       
   13. Add 1 ml PerfectHyb Plus, and shake (80 rpm) for 5 min at RT.
       
   14.  Remove the solution and add 1 ml of fresh PerfectHyb Plus. Shake (80 rpm) at 50 °C in hybridization oven (incubator with rotary shaker can be used) for more than 1 h.
       
   15. Add 1 µl of 10 µM Alexa Fluor-488-labeled oligo d(T). Shake (80 rpm) at 50 °C overnight. It is preferred to perform fluorescent microscopy immediately after the preparation of samples, but the samples can be kept at least for a couple of days at RT, shaded.
       
   
   
2. Microscopic observation
   
   1. Mount the sample in PerfectHyb Plus on slide glass with cover slip.
      
   2. Observe the localization of mRNA with confocal laser-scanning microscopy. Use appropriate setting for the detection of Alexa Fluor-488 (excitation peak 490 nm, emission peak 525 nm). We generally use 488-nm excitation source, and Alexa Fluor-488 fluorescence is recorded between 500 and 600 nm.
      
      
      Figure 2. Distribution of Poly (A) RNA in mesophyll cells of control (TRV-infected) or NbNup75-silenced Nicotiana benthamiana. In NbNup75-silenced plant, abnormal accumulation of mRNA in nuclei is observed as a result of impaired export of mRNA. Bars=30 µm. (Ohtsu et al. 2014)
      

Recipes

1. Fixation cocktail (x 4)
   240 mM NaCl
   14 mM Na₂HPO₄
   6 mM NaH₂PO₄
   5.4 mM KCl
   160 mM EGTA
   Autoclaved and stored at RT
   
2. Fixation solution A (for 10 ml)
   Fixation cocktail (x 4) 2.5 ml
   Formaldehyde 250 µl
   DMSO 500 µl
   Tween20 10 µl
   Heptan 5 ml
   H₂O 1.74 ml
   Prepare before use
   
3. Fixation solution B (for 10 ml)
   Fixation cocktail (x 4) 2.5 ml
   DMSO 500 µl
   Tween 20 10 µl
   Heptan 5 ml
   H₂O 1.99 ml
   Prepare before use

Acknowledgments

This protocol was adapted from Parry et al. (2006) and Germain et al. (2010). The work was supported by a Grant-in-Aid for Scientific Research (B) (26292024) from the Japan Society for the Promotion of Science and by Grant for Basic Science Research Projects from the Sumitomo Foundation.

References

1. Germain, H., Qu, N., Cheng, Y. T., Lee, E., Huang, Y., Dong, O. X., Gannon, P., Huang, S., Ding, P., Li, Y., Sack, F., Zhang, Y. and Li, X. (2010). MOS11: a new component in the mRNA export pathway. PLoS Genet 6(12): e1001250.
   
2. Ohtsu, M., Shibata, Y., Ojika, M., Tamura, K., Hara-Nishimura, I., Mori, H., Kawakita, K. and Takemoto, D. (2014). Nucleoporin 75 is involved in the ethylene-mediated production of phytoalexin for the resistance of Nicotiana benthamiana to Phytophthora infestans. Mol Plant Microbe Interact 27(12): 1318-1330.
   
3. Parry, G., Ward, S., Cernac, A., Dharmasiri, S. and Estelle, M. (2006). The Arabidopsis SUPPRESSOR OF AUXIN RESISTANCE proteins are nucleoporins with an important role in hormone signaling and development. Plant Cell 18(7): 1590-1603.
   
4. Zhang, H. and Liu, Y. (2014). VIGS assays. Bio-protocol 4(5): e1057.